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CELLULAE  TOXINS 


OB   THE 


CHEMICAL  FACTORS 


IN  THE 


CAUSATION  OF  DISEASE 


BY 

VICTOR   C.   VAUGHAN,  M.D.,  LL.D. 

PROFESSOR  OF  HYGIK>T;  AND  PHYSIOLOGICAL  CHEMISTRY  AND  DIRECTOR  OF  THE  HYGIEMIC 
LABORATORY  IN  THE  rNlVERSlTY  OF  MICHIGAN, 


FREDERICK   G.   NOVY,  M.D.,  Sc.D. 

JUNIOR  PROFESSOR  OF  HYGIENE  AND  PHYSIOLOGICAL  CHEMISTRY  IN  THE  UNTTEHSITY 

OF  MICHIGAN. 


FOURTH    EDITION,  REVISED  AND    ENLARGED 


LEA    BEOTHEES    &    COMPANY 

PHILADELPHIA  AND   NEW  YORK 

1902 


Entered  according  to  the  Act  of  Congress  in  the  year  1902,  by 

LEA  BROTHERS  &  CO., 
In  the  Office  of  the  Librarian  of  Congress.    All  rights  reserved. 


TO 
ALBERT   B.    PEESCOTT,  M.D.,  LL.D. 

DIRECTOB  OF  THE  CHEMICAL  LABOBATOEY  IN  THE  UNTVEESITY  OF  MIOHIGAM, 
THIS  LITTLE  WOKK 

IS  EESPECTFULLY  DEDICATED 

AS  A  SLIGHT  TOKEN  OF  THE  HIGH  ESTEEM   IN  WHICH 
HE  IS  HELD  BY  HIS  FORMEK  STUDENTS, 

TELE  AUTHOES. 


PREFACE  TO  FOURTH  EDITION. 

During  the  fourteen  years  which  have  elapsed  since  the  appear- 
ance of  the  first  edition  of  this  book,  the  subject  matter  of  which  it 
treats  has  increased  in  importance,  has  modified  our  conceptions  of 
disease,  and  has  furnished  facts  which  are  now  utilized  in  treatment. 
Quite  naturally,  during  the  growth  and  development  of  the  chem- 
istry of  the  infectious  diseases,  this  science  has  from  time  to  time 
changed  the  relative  importance  of  different  phenomena.  When  the 
first  edition  of  this  book  was  written  it  was  believed  by  those  most 
competent  to  speak  on  the  subject,  that  the  basic  products  of  bacte- 
rial growth  constituted  the  chief  factors  in  the  causation  of  the  in- 
fectious diseases,  but  it  has  been  shown  by  subsequent  discoveries 
that  this  conception  is  erroneous,  and  we  now  look  for  the  specific 
bodies  among  the  synthetic  substances  formed  within  the  cells  of  the 
microorganism.  This  advance  in  knowledge  has  rendered  the  chief 
title  selected  for  former  editions  inappropriate,  and  accounts  for  the 
change  which  we  have  made  therein.  The  text  has  for  the  most  part 
been  rewritten  with  the  intention  of  curtailing  the  space  given  to 
subjects  which  advanced  knowledge  has  shown  to  be  less  important, 
and  also  for  the  purpose  of  introducing  new  matter.  We  regret  ex- 
ceedingly that  want  of  space  has  compelled  us  to  omit  altogether  the 
bibliography  found  in  previous  editions.  References  to  the  litera- 
ture employed  in  the  older  editions  have  been  omitted,  while  those 
bearing  upon  investigations  which  have  been  made  since  the  appear- 
ance of  the  last  edition  are  given  in  footnotes.  Those  to  whom  the 
third  edition  is  accessible  will  be  able  to  look  up  every  piece  of  work 
referred  to  in  this  volume  and  satisfy  themselves  concerning  the  in- 
terpretation which  we  have  placed  upon  the  original  contributions. 
In  order  to  abbreviate  as  much  as  possible,  we  have  omitted  many 
details  given  in  the  previous  editions.  This  curtailment  has  of 
necessity  not  been  uniform  throughout  the  book ;  certain  chapters 
having  been  cut  down  much  more  than  others.  In  some  instances 
the  desire  to  take  up  less  space  has  possibly  led  us  to  omit  state- 
ments of  considerable  importance.  Especially  is  this  true  of  the 
chapter  devoted  to  poisonous  foods.     Several  new  chapters   have 


VI  PREFACE  TO  FOURTH  EDITION. 

been  added,  bringing  into  the  volume  subjects  which  were  wholly 
unknown  at  the  writing  of  the  last  edition.  We  have  endeavored  to 
present  to  our  readers  everything  of  importance  done  in  the  lines 
treated  of  down  to  the  close  of  the  year  1901.  If  this  volume  meets 
with  the  kind  reception  extended  to  its  predecessors,  its  authors  will 
feel  themselves  amply  repaid  for  the  labor  that  they  have  placed 
upon  it. 

Univeksity  of  Michigan,  June  1,  1902. 


CONTENTS. 

CHAPTER   I. 

The  Etiology  of  the  Bacterial  Diseases  :  Bacterial,  Protozoal,  Para- 
sitic, Toxic,  Traumatic,  and  Autogenous  Diseases.  How  Bac- 
teria cause  Disease 17 

CHAPTER   II. 

Classification  and  Definition  of  the  Chemical  Products  of  Bacteria  : 
Ptomains ;  Bacterial  Proteids ;  Bacterial  Cellular  Proteids ; 
Toxins 29 

CHAPTER    III. 
A  Historical  Sketch  of  the  Bacterial  Poisons  :  Observations  on  Putre- 
factive Material ;  Examination  of  Mixed  Cultures  ;  The  Study  of 
Pure  Cultures 38 

CHAPTER   IV. 

The  Bacterial  Poisons  of  some  of  the  Infectious  Diseases ;  Anthrax  ; 
Asiatic  Cholera  ;  Tetanus  ;  Diphtheria  ;  Tuberculosis  ;  Suppura- 
tion ;  Gonorrhoea  ;  the  Summer  Diarrhoeas  of  Infancy  ;  Typhoid 
Fever  ;  Hog  Cholera  ;  Rabbit  Septicaemia  ;  Pneumonia  ;  Malig- 
nant (Edema  ;  Puerperal  Fever  ;  Glanders 48 

CHAPTER   V. 
The  Germicidal  Properties  of  Blood  Serum  ;  Alexins  ;  Nuclein 102 

CHAPTER  VI. 
The  Specific  Precipitins 113 

CHAPTER  VII. 

The   Lysins  :    Bacteriolysis  ;    Hemolysis  ;  Cytolysis  ;    Epitheliolysis  ; 

Nephrolysis  ;  Spermotolysis 122 

CHAPTER  VIII. 

The  Agglutinins 152 

CHAPTER  IX. 

Immunity  :  Natural  Immunity  ;  Natural  Immunity  to  Bacteria  ; 
Natural  Immunity  to  Toxins  ;  Acquired  Immunity  ;  Acquired 
Bacterial  Immunity  ;  Acquired  Immunity  to  Toxins  ;  Antitoxins..  162 

vii 


18  THE  ETIOLOGY  OF  THE  BACTERIAL  DISEASES. 

Some  species  of  single-celled  animal  organisms,  known  as  pro- 
tozoa, may  invade  the  body  and  there  live  and  reproduce  themselves, 
modifying,  impairing  and  destroying  normal  tissue.  The  disorders 
resulting  from  these  causes  are  known  as  protozoal  diseases. 

Other  more  highly  developed  animals  pass  at  least  a  portion  of 
their  lives  as  parasites,  and  we  must  recognize  certain  diseases  as  due 
to  animal  parasites. 

The  living  cells  of  the  animal  body  may  be  altered  or  destroyed 
by  the  action  of  poisons  of  mineral,  vegetable  or  animal  origin. 
The  poisoning  that  results  in  this  way  may  be  acute  or  chronic ;  it 
may  manifest  itself  in  one  case  principally  by  its  action  on  the  nerv- 
ous system,  and  in  another  the  symptoms  induced  may  be  referred 
more  especially  to  the  digestive  organs.  Diseases  due  to  the  ad- 
ministration of  poisons  generated  wholly  outside  the  body  are  grouped 
together  under  the  name  of  intoxicatioiis. 

A  given  group  of  cells  in  the  body  may  be  so  altered  by  mechan- 
ical violence  that  the  continued  performance  of  healthy  function  is 
no  longer  possible.  A  depression  of  the  skull,  as  the  result  of  a  fall 
or  blow,  may  induce  epilepsy  or  insanity.  Diseases  induced  in  this 
manner  are  said  to  be  traumatic. 

Lastly,  without  outside  interference,  any  group  of  cells  in  the 
body  may,  from  having  an  excess  of  work  thrown  on  it,  or  from 
other  causes,  many  of  which  remain  unknown,  fail  to  do  its  duty, 
and,  as  a  consequence,  disaster  may  threaten  the  whole.  These 
diseases  may  be  denominated  as  autogenous. 

This  gives  us  a  simple  etiological  classification  of  diseases  into  : 
(1)  Bacterial,  (2)  fungous,  (3)  protozoal,  (4)  animal  parasitic,  (5) 
intoxications,  (6)  traumatic,  (7)  autogenous. 

While  the  above  given  etiological  classification  of  diseases  is  ad- 
missible, it  must  be  understood  that  in  many  instances  the  cause  is 
not  single,  but  multiple,  and  for  this  reason  sharp  lines  of  classifi- 
cation cannot  be  drawn  ;  for  instance,  the  greatest  danger  in  those 
traumatic  affections  in  which  the  traumatism  itself  does  not  cause 
death,  lies  in  infection.  The  wound  has  simply  provided  a  suitable 
point  of  entrance  for  the  infecting  agent ;  indeed  the  break  in  the  con- 
tinuity of  tissue  may  be  so  slight  that  it  is  of  import  and  danger  only 
on  account  of  the  coincident  or  subsequent  infection,  as  is  true  in 
most  cases  of  tetanus  and  septicemia.  Furthermore,  an  infectious 
disease,  whether  it  originates  in  a  traumatism  or  not,  is  markedly 
influenced  by  what  we  are  pleased  to  call  the  idiosyncrasy  of  the 
patient,  and  by  this  we  mean  the  peculiarities  of  tissue  metabolism 
taking  place  in  the  individual  at  the  time.  A  dozen  men  may  be 
alike  exj)osed  to  the  same  infection,  and  the  infecting  agent  may  find 
a  suitable  soil  for  its  growth  and  development  in  two  of  these,  while 
in  the  other  ten  this  same  agent  meets  with  such  adverse  influences 
that  it  dies  without  producing  any  appreciable  effects ;  or  all  may  be 


ETIOLOGY.  19 

infected,  but  with  difterences  in  degree,  as  is  evidenced  by  variation 
in  symptoms,  in  the  length  of  time  that  the  infecting  agent  con- 
tinues to  grow  and  develop  in  the  body,  and  in  the  ultimate  result. 
Every  physician  who  has  had  experience  in  the  treatment  of  any  of 
the  infectious  diseases  appreciates  the  importance  of  the  personal 
equation  in  his  patients.  It  is  a  fact,  frequently  observed  clinically, 
and  capable  of  experimental  demonstration,  that  privation  and  ex- 
haustion not  only  increase  susceptibility  to  infectious  diseases  but 
also  heighten  mortality  from  the  same. 

That  some  neurotic  affections  originate  from  traumatism,  has  been 
abundantly  demonstrated  ;  that  some  are  largely  due  to  malnutrition 
accompanied  by  improper  metabolism  or  insufficient  elimination,  or, 
in  other  words,  are  to  some  extent  autogenous,  all  believe.  With  a 
clear  understanding  that  the  above  classification  does  not  attempt  a 
sharp  and  marked  differentiation  of  the  causes  of  disease,  we  will 
give  our  attention  to  a  consideration  of  the  etiology  of  the  infectious 
diseases,  and  of  the  traumatic  and  autogenous,  in  so  far  as  these  are 
influenced  by  infection. 

Recognizing  the  fact  that  germs  do  bear  a  causal  relation  to  some 
diseases,  the  question  arises,  how  do  these  organisms  act?  Inas- 
much as  anthrax  was  the  first  disease  demonstrated  to  be  due  to  bac- 
teria, attempts  to  answer  this  question  have  generally  been  made  by 
reference  to  the  microorganism  of  this  affection,  or,  in  other  words, 
the  question  is  changed  so  as  to  read,  "  In  what  way  does  the  bacillus 
anthracis  induce  the  symptoms  of  this  disease  and  cause  death  ? " 
Of  the  proposed  solutions  of  this  problem  the  following  are  the  most 
important  : 

1.  It  was  suggested  by  Bollinger  that  apoplectiform  anthrax  is 
due  to  deoxidation  of  the  blood  by  the  bacilli.  These  germs  are 
aerobic,  and  it  was  thought  that  they  might  act  by  depriving  the  red 
blood  corpuscles  of  their  oxygen.  This  theory  was  rendered  more 
tenable  by  the  resemblance  of  the  symptoms  of  anthrax  to  those  of 
carbonic  acid  poisoning.  The  most  prominent  of  these  symptoms 
are  dyspnoea,  cyanosis,  convulsions,  dilated  pupils,  subnormal  tem- 
perature, and,  in  general,  the  phenomena  of  asphyxia.  Moreover, 
post-mortem  examination  reveals  conditions  similar  to  those  observed 
after  death  by  deprivation  of  oxygen  ;  the  veins  are  distended,  the 
blood  is  dark  and  thick,  the  parenchymatous  organs  are  cyanotic, 
and  the  lungs  are  hyperemic.  Apoplectiform  anthrax  was  compared 
to  poisoning  with  hydrocyanic  acid,  which  was  at  one  time  believed 
to  produce  fatal  results  by  robbing  the  blood  of  its  oxygen. 

This  theory  presupposed  a  large  number  of  bacilli  in  the  blood, 
and  this  accorded  with  the  estimate  of  Davaine,  which  placed  the 
number  at  from  eight  to  ten  million  in  a  single  drop ;  but  more  ex- 
tended and  careful  observation  showed  that  the  blood  of  animals 
dead  from  anthrax  is  often  very  poor  in  bacilli.     Virchow  reported 


20  THE  ETIOLOGY  OF  THE  BACTERIAL  DISEASES. 

cases  of  this  kind  and  Bollinger  himself  found  the  bacilli  often  con- 
fined to  certain  organs,  and  not  abundant  in  the  blood.  Later, 
Siedamgrotzky  counted  the  organisms  in  the  blood  in  various  cases 
and  found  not  only  that  the  estimate  made  by  Davaine  is  too  large, 
but  that  in  many  instances  the  number  present  in  the  blood  is  small, 
while  Joffroy  observed  in  some  of  his  inoculation  experiments  that 
the  animals  died  before  any  bacilli  appeared  in  the  blood.  These 
and  other  investigations  of  similar  character  caused  workers  in  this 
field  of  research  to  doubt  the  truth  of  the  theory  of  Bollinger,  and 
these  doubts  were  soon  converted  into  positive  evidence  against  it ; 
but  for  a  while  it  was  the  subject  of  an  interesting  controversy. 
Pasteur,  in  support  of  the  theory,  reported  that  birds  were  not  sus- 
ceptible to  anthrax,  and  he  accounted  for  this  by  supposing  that 
the  blood  corpuscles  in  birds  do  not  part  with  their  oxygen  readily. 
However,  it  was  shown  by  Oemler  and  Feser  that  the  learned 
Frenchman  had  generalized  from  limited  data,  and  that  many  birds 
are  especially  susceptible  to  this  disease.  Oemler  found  that  the 
blood,  even  when  rich  in  anthrax  bacilli,  still  possesses  the  bright 
red  color  of  oxyhemoglobin.  Toepper  and  Roloff  reported  cases  of 
apoplectiform  anthrax  in  which  there  was  no  difficulty  in  respiration, 
and  Toussaint  caused  animals  which  had  been  inoculated  with  the 
anthrax  bacillus  to  breathe  air  containing  a  large  volume  of  oxygen 
and  found  that  this  did  not  modify  the  symptoms  or  retard  death. 
Finally,  Nencki  determined  the  amount  of  physiological  oxidation 
going  on  in  the  bodies  of  animals  sick  with  anthrax  by  estimating 
the  amount  of  phenol  excreted  after  the  administration  of  one  gram 
of  benzol,  and  found  that  the  oxidation  of  the  benzol  was  not  dimin- 
ished by  the  disease.  In  short,  the  theory  that  germs  destroy  life 
by  depriving  the  blood  of  its  oxygen  has  been  found  not  to  be  true 
for  anthrax,  and  if  not  true  for  anthrax,  certainly  it  cannot  be  for 
any  other  known  disease.  The  bacillus  anthracis  is,  as  has  been 
stated,  aerobic,  while  many  of  the  pathogenic  bacteria  are  anaerobic 
— that  is,  they  live  in  the  absence  of  oxygen  ;  this  element  is  not 
necessary  to  their  existence,  and  indeed,  when  present  in  large 
amount,  it  is  fatal  to  them.  Moreover,  in  many  diseases  the  bacteria 
are  not  found  in  the  blood  at  all,  and,  lastly,  the  symptoms  of  these 
diseases  are  not  those  of  asphyxia.  These  facts  have  caused  a  com- 
plete abandonment  of  this  theory. 

2.  If  a  properly  stained  section  of  a  kidney  taken  from  a  guinea- 
pig,  which  has  been  inoculated  with  the  bacillus  anthracis,  be  ex- 
amined under  the  microscope,  the  bacilli  will  be  found  to  be  pres- 
ent in  such  large  numbers  that  they  form  emboli,  which  not  only 
close,  but  actually  distend  the  capillaries  and  even  larger  blood  ves- 
sels, and  interfere  with  the  normal  functions  of  the  organ.  A 
similar  condition  is  sometimes  found  on  microscopical  examination  of 
the  liver,  spleen   and  lungs.     From   these  appearances  it  was  in- 


ETIOLOGY,  21 

ferred  by  Bollinger  that  the  bacilli  produce  the  diseased  condition 
by  accumulating  in  large  numbers  in  these  important  organs,  and 
mechanically  interrupting  their  functions.  This  is  known  as  the 
mechanical  interference  theory. 

If  anthrax  were  the  only  infectious  disease,  or  if  in  other  infec- 
tions the  germs  were  as  numerous  in  the  blood  as  they  are  in  anthrax, 
the  mechanical  interference  theory  would  still  have  strong  support, 
but  to  the  majority  of  germ  diseases  it  is  not  at  all  applicable. 

3.  Another  answer  given  to  this  question,  "  How  do  germs  cause 
disease?"  is  that  they  do  so  by  consuming  the  proteids  of  the  body, 
and  thus  depriving  it  of  its  sustenance.  The  proteids  are  known  to 
be  necessary  for  the  building  up  of  cells  and  it  is  also  known  that 
microorganisms  feed  upon  proteids.  However,  this  theory  is  unten- 
able for  several  reasons  :  in  the  first  place,  many  of  the  infectious  dis- 
eases destroy  life  so  quickly  that  the  fatal  effects  cannot  be  supposed 
to  be  due  to  the  consumption  of  any  large  amount  of  proteid ;  in  the 
second  place,  the  distribution  of  the  microorganisms  is  such  that 
they  do  not  come  in  contact  with  any  large  proportion  of  the  pro- 
teids of  the  body ;  in  the  third  place,  the  symptoms  of  the  majority 
of  the  bacterial  diseases  are  not  those  which  would  be  produced  by 
withdrawing  from  the  various  organs  their  food.  The  symptoms  are 
not  those  of  starvation. 

4.  Still  another  theory,  which  has  been  offered,  is  that  the 
bacteria  destroy  the  blood  corpuscles,  or  lead  to  their  rapid  dis- 
integration. But  in  many  of  the  infectious  diseases,  as  has 
been  stated,the  microorganisms,  although  abundant  in  some  or- 
gans, are  not  present  in  the  blood.  Moreover,  the  disintegration 
of  the  blood  corpuscles  is  not  confirmed  by  microscopical  exami- 
nation. 

5.  Seeing  the  vital  deficiencies  in  the  above  theories,  and  being 
impressed  by  the  results  obtained  by  the  chemical  study  of  putrefac- 
tion, bacteriologists  have  been  led  to  inquire  into  the  possibility 
of  the  symptoms  of  the  infectious  diseases  being  due  to  chemical 
poisons.  In  investigating  this  theory,  the  following  possibilities 
suggest  themselves : 

(a)  The  microorganisms  may  be  intimately  associated  with,  or 
may  produce,  a  soluble  chemical  ferment,  which  by  its  action  on  the 
body  produces  the  symptoms  of  the  disease  and  death.  At  one  time 
this  theory  had  a  number  of  ardent  supporters,  among  whom  might 
be  mentioned  the  eminent  scientist  De  Bary ;  but  Pasteur  proved 
the  theory  false  when  he  filtered  anthrax  blood  through  earthen 
cylinders,  inoculated  animals  with  the  filtrate  and  failed  to  pro- 
duce any  effect.  Nencki  made  a  similar  demonstration  when  he 
inoculated  a  two  per  cent,  gelatin  preparation  with  the  anthrax 
bacillus,  which  liquefied  the  gelatin,  and  on  standing  the  bacilli 
settled  to  the  bottom,  after  which  the  supernatant  fluid  which  was, 


22  THE  ETIOLOGY  OF  THE  BACTERIAL  DISEASES. 

clear  and  alkaline  in  reaction,  was  filtered  and  injected  into  animals 
without  producing  any  eflPect. 

It  must  not  be  inferred  from  the  above  statements  that  bacteria 
do  not  produce  ferments.  Many  of  them  do  form  both  diastatic  and 
peptic  ferments,  which  may  retain  their  activity  after  the  bacteria 
have  been  destroyed ;  but  there  is  no  proof  in  any  case  that  these 
ferments  have  a  causal  relation  to  the  disease.  After  the  disease 
process  has  been  inaugurated  some  of  these  ferments  probably  play 
an  important  part  in  the  production  of  morphological  changes,  the 
nature  of  which  will  be  indicated  when  the  different  diseases  are  dis- 
cussed. 

(6)  The  microorganisms  may  act  either  directly  or  indirectly  as 
ferments,  spHtting  up  complex  proteids  in  the  tissue  and  producing 
among  these  split  products  the  specific  poisons  which  induce  the 
characteristic  symptoms  of  the  disease,  and  may  cause  death.  This 
theory,  once  quite  generally  held,  has  stimulated  numerous  investi- 
gations, some  of  which  have  led  to  important  discoveries ;  but  at 
present  it  is  safe  to  say  that  among  the  bacterial  split  products  formed 
either  in  artificial  culture  media  or  in  the  body,  there  is  not  found 
one  which,  on  account  of  its  intensity  of  action  or  from  the  nature  of 
the  symptoms  which  it  produces,  can  be  regarded  as  the  specific  cause 
of  any  one  of  the  infectious  diseases.  Moreover,  it  has  been  shown 
that  some  of  the  most  virulent  germs,  as  for  instance,  the  bacillus  of 
tetanus,  will  grow  and  retain  their  virulence  in  artificial  cultures 
made  up  principally  of  inorganic  substances  and  containing  only 
minute  quantities  of  organic  bodies  of  such  simple  construction  that 
it  must  be  admitted  that  the  specific  toxins  of  these  microorganisms 
cannot  result  from  their  cleavage  action. 

While  we  are  forced  to  conclude  that  no  specific  toxin  has  been 
found  among  the  cleavage  products  of  bacteria,  it  is  well  established 
that  certain  powerful  poisons  originate  in  this  way,  and  it  will  come 
within  the  scope  of  this  treatise  to  deal  with  all  substances  formed  by 
the  cleavage  action  of  bacteria,  both  upon  the  constituents  of  artificial 
culture  media  and  within  the  animal  body. 

(c)  Poisons  may  be  produced  by  the  cellular  activity  of  bacteria 
much  in  the  same  way  as  morphin  is  formed  in  the  poppy.  This 
theory  supposes  that  the  formation  of  bacterial  toxins  is  a  synthetical, 
rather  than  an  analytical,  process.  It  is  now  generally  believed  that 
most,  if  not  all,  of  the  pathogenic  microorganisms  consist  of  cell  walls 
containing  cell  protoplasm,  and  that  the  specific  toxin  is  a  constit- 
uent of  the  protoplasm,  and  that  its  formation  is  one  of  the  vital 
phenomena  manifested  by  the  organism  in  its  processes  of  growth 
and  multiplication.  In  some  species  the  cell  wall  is  not  easily  per- 
meable and  the  toxin  is  found  only  within  the  cell ;  while  in  other 
species  the  toxin  formed  within  the  cell  readily  passes  through  the 
wall  and  diffuses  through  the  culture  media  in  artificial  growths,  or 


ETIOLOGY.  23 

through  the  tissues,  when  the  germ  is  multiplying  in  the  animal 
body.  In  at  least  some  species  the  formation  of  a  toxin  is  not  a 
phenomenon  which  invariably  accompanies  growth  and  multipli- 
cation. This  is  shown  to  be  true  by  the  frequently  observed  fact 
that  a  highly  virulent  germ  may  under  certain  conditions  wholly 
lose  its  toxicity  while  it  continues  to  vegetate  most  luxuriantly.  It 
seems  to  be  evident  that  certain  conditions  of  growth,  as,  for  in- 
stance, the  nature  of  the  medium,  the  temperature,  the  supply  of 
oxygen,  and  the  presence  or  absence  of  certain  chemical  agents,  de- 
termine the  amount  of  specific  toxin  formed  within  the  cell.  Most 
pathogenic  germs  find  the  conditions  suitable  for  the  elaboration 
of  poisons  at  their  optimum  in  the  animal  body,  and  for  this  reason 
their  virulence  is  increased  by  passing  successively  through  a  series 
of  animals.  However,  this  is  not  always  true,  and  germs  may  de- 
crease in  virulence  by  being  passed  through  certain  animals ;  some 
observers  have  reported  the  finding  of  certain  microorganisms  that 
invariably  decrease  in  virulence  on  being  transferred  from  one 
animal  to  another.  There  are  many  interesting  questions  along  this 
line  which  need  to  be  investigated  much  more  fully  than  has  been 
done  up  to  the  present  time  before  we  can  speak  positively  concern- 
ing them.  They  furnish  the  basis  of  problems  that  are  of  both 
scientific  and  practical  interest.  In  many  epidemics  the  specific 
microdrganism,  to  which  the  epidemic  disease  is  due,  apparently  in- 
creases for  a  while  in  virulence  and  then  gradually  seems  to  become 
less  dangerous.  However,  a  discussion  of  these  questions  would  take 
us  too  far  away  from  the  subject  matter  just  now  in  hand. 

We  will  now  give  what  appears  to  us,  in  the  present  state  of  our 
knowledge,  a  correct  definition  of  an  infectious  disease  : 

An  infectious  disease  arises  when  a  specific,  pathogenic  microor- 
ganism, having  gained  admittance  to  the  body  and  having  found  con- 
ditions favorable,  grows  and  multiplies,  and  in  so  doing  elaborates  a 
chemical  poison  which  induces  its  characteristic  effects. 

In  the  systemic  infectious  disease,  such  as  anthrax,  typhoid  fever 
and  cholera,  the  specific  poison  is  undoubtedly  taken  into  the  gen- 
eral circulation,  and  may  reach  and  influence  every  part  of  the  body. 
In  the  local  infectious  diseases,  such  as  gonorrhea  and  infectious 
ophthalmia,  the  first  action  of  the  poison  seems  to  be  confined  to  the 
place  of  its  formation  ;  although  even  in  these,  when  of  a  specially 
virulent  type,  the  effects  may  extend  to  the  general  health,  or  the 
poison  may  strongly  act  on  some  distant  part  of  the  body.  It  is 
probably  true  that  in  many  of  the  infectious  diseases  the  chemical 
poison  has  both  a  local  and  a  systemic  action  ;  thus,  it  is  by  no  means 
certain  that  the  ulceration  of  typhoid  fever  is  due  directly  to  the 
living  bacillus,  for  it  is  now  an  established  fact  that  this  disease  may 
exist,  run  a  typical  course,  and  end  in  death,  without  anatomical 
changes   in    the    intestine.     In    diphtheria  and  tetanus  the  toxin 


24  THE  ETIOLOGY  OF  THE  BACTERIAL  DISEASES. 

formed  within  the  bacterial  cells  readily  diffuses  through  the  cell 
walls  and  enters  the  circulation,  while  the  organism  itself  is  confined 
to  relatively  a  small  area  and  may  not  be  found  in  the  blood  at  all. 
Such  diseases  are  properly  called  bacterial  intoxications.  In  some 
other  infectious  diseases,  such  as  anthrax  and  one  form  of  the  plague, 
the  germ  itself  may  be  distributed  by  the  blood  and  lymph  to  every 
part  of  the  body  ;  these  diseases  are  designated  as  septicemias. 

With  the  proof  established  that  the  deleterious  effects  wrought  by 
germs  are  due  to  chemical  poisons  elaborated  by  them,  let  us  inquire 
what  properties  a  microorganism  must  possess  before  it  can  be  said 
to  be  the  specific  cause  of  a  disease.  The  four  rules  of  Koch  have 
been  conceded  to  be  sufficient  to  show  that  a  given  germ  is  the  sole 
and  efficient  cause  of  the  disease  with  which  that  germ  is  associated. 
Briefly  these  rules  are  as  follows  : 

1.  The  germ  must  be  present  in  all  cases  of  that  disease. 

2.  It  must  be  isolated  from  other  organisms  and  from  all  other 
matter  found  with  it  in  the  diseased  animal. 

3.  The  germs  thus  freed  from  all  other  foreign  matter  must,  when 
properly  introduced,  produce  the  disease  in  healthy  animals. 

4.  The  microorganism  must  be  found  properly  distributed  in  the 
animal  in  which  the  disease  has  been  induced. 

We  will  briefly  discuss  the  applicability  of  these  rules.  When  it 
is  stated  that  the  germ  must  be  present  in  all  cases  of  the  disease,  it 
need  not  be  understood  that  an  unlimited  number  of  cases  must  be 
examined  before  the  causal  relation  of  a  given  organism  to  the  disease 
may  be  reasonably  suspected.  This  would  require  more  than  a  life- 
time, and  would  demand  facilities  for  the  study  of  the  special  disease 
that  do  not  and  cannot  exist.  The  number  of  cases  in  which  the  germ 
is  constantly  found  should  be  reasonably  large,  and  the  larger  this 
number  the  greater  the  probability  that  the  organism  is  etiologically 
related  to  the  disease.  Moreover,  the  germ  may  be  present  in  all 
cases,  and  yet  it  may  not  be  found  in  all.  To  demand  that  it  be 
found  in  all  cases  would  be  to  presume  that  the  methods  of  detecting 
and  recognizing  a  given  organism  are  perfect,  and  there  is  no  ground 
for  this  assumption.  Again,  since  the  results  of  no  one  man's  work 
can  be  accepted  in  science  until  they  have  been  confirmed  by  others, 
the  personal  equation  must  be  considered ;  what  one  man  finds, 
another  may  fail  to  find.  Diligence,  skill  and  accuracy  are  not 
equally  developed  in  all  men,  and,  moreover,  the  methods  employed 
may  differ.  To  illustrate  these  points  :  Koch,  after  the  most  pains- 
taking study  embracing  twenty-nine  cases  of  pulmonary  tuberculosis, 
nineteen  of  miliary  tuberculosis,  twenty -one  of  tuberculous  glands, 
thirteen  of  tuberculous  joints,  ten  of  tuberculosis  of  the  bones,  four 
of  lupus,  and  seventeen  of  bovine  tuberculosis,  announced  that  he 
had  discovered  a  bacillus  which  is  constantly  present  in  tubercular 
disease.     Since  this  announcement  thousands  of  physicians  and  bac- 


ETIOLOGY.  25 

■  teriologists,  possessing  different  degrees  of  skill,  and  often  by  differ- 
ent methods  of  staining,  with  microscopes  of  all  kinds,  good  and  bad, 
have  sought  for  this  bacillus,  and  it  is  not  strange  that  now  and  then 
some  man  fails  to  find  the  organism  in  a  genuine  case  of  tuberculosis. 

Another  most  important  point  in  this  connection  lies  in  the  fact 
that  the  clinical  and  the  bacteriological  diagnoses  do  not  always  agree. 
The  most  skilful  clinicians  may  differ  concerning  a  case  of  membra- 
nous sore  throat.  One  is  sure  that  it  is  diphtheria ;  a  second  is  in 
doubt  and  reports  it  as  a  suspicious  or  doubtful  case ;  and  a  third  is 
sure  that  it  is  not  diphtheria.  A  bacteriological  examination  may 
reveal  or  fail  to  reveal  the  presence  of  the  Loeffler  bacillus.  Again, 
it  may  be  that  any  number  of  the  most  competent  clinicians  agree 
in  saying  that  the  case  is  or  is  not  one  of  diphtheria,  and  yet  a  bac- 
teriological examination  may  result  in  a  contradictory  diagnosis. 
This  is  exactly  what  has  happened  in  the  study  of  diphtheria.  From 
statistics  gathered  by  Novy,  it  appears  that  of  8,186  cases  of  clinical 
diphtheria,  diagnosed  as  such  by  different  men  in  Europe  and  Amer- 
ica (to  May,  1895),  the  Loeffler  bacillus  was  found  in  5,943,  the 
bacteriological  examinations  also  being  made  by  different  men.  The 
clinical  diagnosis  was  confirmed  bacteriologically  in  72.6  per  cent,  of 
these  cases.  On  the  other  hand,  of  333  cases  diagnosed  as  diphtheria 
by  Baginsky,  332  furnished  the  bacillus,  and  of  117  seen  by  Kossel, 
all  were  confirmed  by  the  bacteriological  examination.  These  figures 
are  given  to  illustrate  the  factors  of  variation  that  may  arise  in  the 
application  of  the  first  of  Koch's  rules. 

Shall  we  accept  the  clinical  or  the  bacteriological  classification  of 
disease  ?  There  can  be  no  doubt  that  the  latter  is  the  more  exact, 
and  its  adoption  will  lead  to  a  more  accurate  and  scientific  study  of 
disease.  An  etiological  classification  of  the  infectious  diseases  is  one 
of  the  great  desiderata  of  scientific  medicine.  Whether  it  will  ulti- 
mately be  made  upon  the  morphological  characters  of  the  bacteria  or 
on  their  poisonous  products  cannot  yet  be  determined.  There  are 
certain  objections  to  making  the  first  of  these  the  basis  that  seem 
well-nigh  insuperable,  and  some  of  these  will  be  discussed  later. 

The  importance  of  the  first  of  Koch's  rules  is  self-evident ;  how- 
ever, the  invariable  presence  of  any  germ  in  a  certain  disease  does 
not  prove  that  the  former  is  the  cause  of  the  latter.  Indeed,  so  long 
as  the  investigation  goes  no  further  than  this,  we  are  justified  in  say- 
ing that  the  microorganism  may  be  an  accompaniment  or  a  conse- 
quence of  the  disease  ;  therefore,  additional  evidence  is  wanting,  and 
is  furnished  by  complying  with  the  other  rules  of  Koch. 

The  second  rule  is  complied  with  by  means  of  plate  and  other  cul- 
tures, a  description  of  which  would  be  out  of  place  here. 

The  third  and  fourth  rules  are  difficult  of  application,  because  the 
lower  animals  are  often  immune  to  many  of  the  diseases  to  which 
man  is  susceptible. 


26  THE  ETIOLOGY  OF  THE  BACTERIAL  DISEASES. 

The  time  will  probably  come  when  the  true  test  of  the  specific 
character  of  a  germ  will  be  made  with  the  chemical  products.  A 
given  bacterium  may  not  multiply  in  the  circulating  blood  of  a  dog, 
and  failure  to  do  so  is  by  no  means  proof  that  the  same  organism 
might  not  cause  disease  in  man  ;  but  every  germ  which  causes  dis- 
ease in  man  does  so  by  its  chemical  products  ;  and  if  these  be  isolated 
and  injected  into  the  dog  in  sufficient  quantity  a  poisonous  effect  will 
most  likely  follow.  In  the  study  of  the  bacteria  of  the  infectious 
diseases,  the  third  and  fourth  of  Koch's  rules  have  not  been  com- 
plied with  in  many  cases,  as  has  been  stated,  on  account  of  the  in- 
susceptibility of  the  lower  animals.  The  majority  of  investigators, 
meeting  with  this  difficulty,  have  been  inclined  to  rest  content  with 
the  first  two  rules,  and  to  conclude  that  when  a  given  germ  is  con- 
stantly present  in  a  given  disease  and  not  found  in  other  diseases, 
that  it  is  the  cause  of  the  disease  with  which  it  is  associated.  In 
1889,  Vaughan  suggested  that  in  those  instances  in  which  the  third 
and  fourth  of  Koch's  rules  cannot  be  complied  with  on  account  ot 
the  insusceptibility  of  the  lower  animals,  it  should  be  shown  that 
the  germ  can  produce  chemical  poisons  which  will  induce  in  the 
lower  animals  in  an  acute  form  the  characteristic  symptoms  of  the 
disease,  before  the  proof  that  the  given  germ  is  the  cause  of  the  dis- 
ease be  accepted  as  positive. 

The  science  of  bacteriology  has  of  necessity  been  largely  founded 
upon  morphological  studies.  Bacteriologists  have  given  their  time 
and  attention  to  the  discovery  of  bacterial  forms  in  the  diseased 
organism,  and  to  observations  of  characteristics  in  structure  and 
growth  of  different  species  of  bacterial  life.  This  is  perfectly  proper, 
but  we  must  also  study  the  physiology  and  chemistry  of  germs  and 
until  this  is  done  we  must  remain  ignorant  of  the  true  cause  of  dis- 
ease, and  so  long  as  we  remain  ignorant  of  the  cause  it  cannot  be 
expected  that  we  shall  discover  scientific  and  successful  methods 
of  treatment.  Suppose  that  our  knowledge  of  the  yeast  plant  was 
limited  to  its  form  and  method  of  growth ;  of  how  little  practical 
importance  would  this  knowledge  be.  That  the  yeast  plant  requires 
a  saccharine  soil  before  it  can  grow,  that  given  such  a  soil  it  produces 
carbonic  acid  gas  and  alcohol  are  the  most  important  and  practical 
facts  which  have  been  ascertained  in  its  study.  Likewise,  the  con- 
ditions under  which  pathogenic  germs  multiply,  and  the  products 
which  they  elaborate  in  their  multiplication,  must  be  ascertained  be- 
fore their  true  and  complete  relationship  to  disease  can  be  understood. 

In  saying  that  the  morphological  work  upon  which  the  science  of 
bacteriology  largely  depends  is  inadequate,  we  wish  that  it  may  be 
plainly  understood  that  we  are  not  offering  any  hostile  criticisms 
upon  the  great  men  who  have  done  this  work  and  who  have  formu- 
lated conclusions  therefrom.  The  development  of  bacteriology  has 
been  in  accordance  with  the  natural  law  governing  the  growth  of  all 


ETIOLOGY.  27 

the  biological  sciences.  The  study  of  form  naturally  and  necessarily 
precedes  the  study  of  function.  The  ornithologist  finds  a  new  species 
of  bird ;  he  first  studies  its  shape  and  size,  the  color  of  its  plumage, 
the  form  of  its  beak,  the  number  and  arrangement  of  the  feathers  of 
the  tail  and  wing,  the  color  of  the  eyes,  etc.  All  this  can  be  done 
with  a  single  specimen,  recognizing  the  fact,  however,  that  variations 
more  or  less  marked  are  likely  to  be  found  in  other  individuals. 
More  time  and  wider  opportunities  for  observation  will  be  needed 
before  he  can  tell  where  and  when  this  bird  is  accustomed  to  build 
its  nest,  upon  what  insects,  grains  and  berries  it  feeds,  with  what 
other  species  of  birds  it  lives  in  peace,  and  with  what  it  is  at  war. 
A  much  greater  range  of  observation  and  study  is  necessary  before 
the  naturalist  can  tell  how  his  newly  discovered  species  would  thrive 
if  carried  to  a  new  climate  where  it  would  be  compelled  to  live  upon 
unaccustomed  food,  to  build  its  nest  of  strange  material,  and  to 
encounter  new  foes. 

We  repeat  that  it  is  no  discredit  to  the  science  nor  to  those  who 
have  developed  it  to  say  that  the  study  of  bacteriology  has  largely 
been  morphological.  Without  the  morphologist,  the  physiologist 
and  the  physiological  chemist  could  not  exist.  The  science  having 
had  for  its  support  largely  morphological  studies,  the  deductions 
and  formulated  statements  arrived  at  by  its  students  have  been 
reached  in  accordance  with  the  knowledge  obtained  from  this 
source  ;  but  since  it  has  been  admitted  that  the  causal  relationship 
between  a  given  germ  and  a  certain  disease  is  dependent  upon  the 
chemical  products  of  the  growth  of  the  germ,  the  fundamental  lines 
of  work  must  be  made  to  correspond  with  this  knowledge. 

We  may  briefly  enumerate  some  of  the  problems  that  now  lie  be- 
fore us  in  our  attempts  to  study  the  chemical  factors  in  the  causation 
of  the  bacterial  diseases.  In  the  first  place,  it  will  be  desirable  to 
define  and  classify  the  chemical  products  of  bacterial  activity.  In 
doing  this  it  will  be  desirable  at  all  times  to  distinguish  between 
those  substances  which  have  their  origin  in  the  cleavage  action 
of  bacteria  and  those  which  are  formed  by  synthetical  processes 
within  the  cell ;  but  our  knowledge  upon  this  subject  is  not  as  yet 
sufficiently  advanced,  nor  does  it  possess  that  degree  of  accuracy 
necessary  before  we  can  determine  positively  the  manner  of  formation 
of  these  substances.  In  defining  and  classifying  the  chemical  prod- 
ucts of  bacterial  activity  we  will  find  quite  naturally  that  there  is 
not  complete  agreement  among  those  who  have  worked  in  this  field 
of  science.  Every  attempt  at  the  classification  of  bacterial  products 
must  at  present  be  regarded  as  provisional  and  subject  to  such  alter- 
ations as  future  discoveries  may  indicate.  It  will  also  be  found 
that  it  is  not  possible  in  all  cases  to  distinguish  between  poisonous 
and  harmless  bacterial  products.  This  subject  will  be  discussed  more 
in  detail  later. 


28  THE  ETIOLOGY  OF  THE  BACTERIAL  DISEASES. 

In  studying  bacterial  products  it  will  be  necessary  for  us  to  con- 
sider the  conditions  under  which  they  are  formed.  We  will  be  com- 
pelled to  study  those  substances  formed  in  artificial  cultures  as  well 
as  those  formed  in  the  animal  body.  Indeed,  in  some  instances  we 
will  find  our  knowledge  confined  to  investigations  made  under  more 
or  less  artificial  conditions,  and  it  will  be  well  for  us  to  remember 
that  caution  must  be  exercised  in  drawing  conclusions  from  knowl- 
edge thus  obtained.  It  might  be  well  at  this  place  to  distinguish 
between  pathogenic  and  toxicogenic  bacteria.  A  pathogenic  bac- 
terium is  one  which,  as  the  name  implies,  induces  a  specific  disease 
which  is  recognized  by  more  or  less  well-marked  and  characteristic 
symptoms  during  life,  or  by  more  or  less  definite  lesions  found  after 
death,  or  by  both.  Pathogenic  bacteria  are  always  capable  of  growth 
in  the  animal  body,  in  which  they  multiply  and  elaborate  their  spe- 
cific toxins.  All  pathogenic  germs  are  toxicogenic,  but  it  does  not 
follow  that  all  toxicogenic  bacteria  are  pathogenic.  A  toxicogenic 
microorganism  is  one  which,  as  this  name  also  implies,  is  capable  of 
producing  a  poison  or  poisons.  A  toxicogenic  germ  may  or  may  not 
be  capable  of  growth  in  the  animal  body.  It  may  multiply  in  milk 
or  some  other  article  of  food  before  its  introduction  into  the  body, 
and  may  in  this  menstruum  elaborate  more  or  less  powerful  poisons. 

In  the  second  place  we  will  proceed  to  give  a  brief  historical 
sketch  of  the  discovery  and  study  of  the  bacterial  poisons ;  and, 
thirdly,  we  will  pass  on  to  a  discussion  of  the  special  products  of  the 
specific  pathogenic  bacteria.  In  doing  this  we  will  not  confine  our- 
selves to  the  chemistry  of  toxins  but  will  extend  our  discussion  to 
their  action  on  animals  and  the  pathological  changes  induced  by  them. 

After  discussing  the  subjects  mentioned  above,  we  will  proceed  to 
the  study  of  those  conditions  of  the  animal  body  which  influence  the 
growth  and  development  of  bacteria  in  the  same.  This  will  involve 
a  study  of  the  germicidal  constituents  of  the  blood,  the  agglutinating 
action  of  blood  serum,  the  production  and  action  of  the  toxins,  the 
production  of  immunity  and  the  treatment  of  the  infectious  diseases 
by  serum  therapy. 

On  the  conclusion  of  the  above-mentioned  studies  we  will  investi- 
gate questions  pertaining  to  poisonous  foods  which  owe  their  harm- 
ful properties  to  the  presence  of  toxicogenic  bacteria.  In  this  con- 
nection we  will  present  the  known  facts  concerning  food  poisons  and 
the  methods  of  their  detection. 


CHAPTER    IL 

CLASSIFICATION   AND   DEFINITION   OF   THE    CHEMICAL 
PRODUCTS   OF   BACTERIA. 

Ptomains. — While  an  exact  classification  of  the  chemical  products 
of  bacterial  growth  cannot  be  made  at  present,  we  know  of  two  dis- 
tinct classes,  one  of  which  contains  substances  which  combine  with 
acids  forming  salts,  and  which  in  this  respect  at  least  corresponds 
with  the  inorganic  and  vegetable  bases.  The  members  of  this 
class  are  designated  as  ptomains,  a  name  suggested  by  the  Italian 
toxicologist  Selmi,  and  derived  from  the  Greek  word  -zcofia  meaning 
a  cadaver.  A  ptomain  may  be  defined  as  an  organic  chemical  com- 
pound, basic  in  character,  and  formed  by  the  action  of  bacteria  on 
nitrogenous  matter.  On  account  of  their  basic  properties,  in  which 
they  resemble  the  vegetable  alkaloids,  ptomains  may  be  called  putre- 
factive alkaloids.  They  have  also  been  designated  as  animal  alka- 
loids, but  this  is  a  misnomer,  because,  in  the  first  place,  some  of  them 
are  formed  in  the  putrefaction  of  vegetable  matter  ;  and,  in  the  second 
place,  the  term  "  animal  alkaloid  "  is  more  properly  restricted  to  the 
leucomains — those  basic  substances  which  result  from  tissue  metabo- 
lism in  the  body.  Kobert  employs  the  term  ptomatin  as  etymolog- 
ically  preferable  to  ptomain. 

While  some  of  the  ptomains  are  highly  poisonous,  this  is  not  an 
essential  property,  and  others  are  wholly  inert.  Indeed,  the  greater 
number  of  those  which  have  been  isolated  do  not,  when  employed  in 
single  doses,  produce  any  apparent  harmful  effects.  Brieger  has  sug- 
gested that  the  term  ptomain  be  restricted  to  the  non-poisonous  basic 
products,  and  he  designates  the  poisonous  ones  as  "  toxins  " ;  how- 
ever, such  a  classification  would  be  of  questionable  utility.  It  will 
be  necessary  for  us  to  possess  more  extensive  and  exact  knowledge 
concerning  the  action  of  many  of  these  substances  before  we  can 
decide  positively  which  are  poisonous  and  which  are  not.  The  effect 
of  a  chemical  compound  upon  the  animal  body  depends  upon  the 
conditions  under  w^hich,  and  the  time  during  which  it  is  administered. 
Thirty  grains  of  quinine  may  be  taken  by  a  healthy  man  during 
twenty-four  hours  without  any  appreciable  ill  effect,  yet  few  of  us 
would  be  willing  to  admit  that  the  administration  of  this  amount 
daily  for  months  would  be  wise  or  altogether  free  from  injury.  In 
the  same  manner,  the  administration  of  a  given  quantity  of  a  bacterial 
alkaloid  to  a  dog  or  a  guinea-pig  in  a  single  dose  may  do  no  harm, 
while  the  daily  production  of  the  same  substance  in  the  intestine  of  a 

29 


30  CHEMICAL  PRODUCTS  OF  BACTERIA. 

man  and  its  absorption,  continued  through  weeks  and  possibly  years, 
may  be  of  marked  detriment  to  the  health.  We  are  not  as  yet  in 
possession  of  sufficient  knowledge  concerning  the  physiological  or  tox- 
icological  action  of  the  putrefactive  alkaloids  to  render  the  classifi- 
cation proposed  by  Brieger  worthy  of  general  adoption ;  moreover, 
the  term  "  toxin  "  is  quite  generally,  although  somewhat  incorrectly, 
employed  to  designate  those  non-basic  bacterial  poisons  for  which 
Brieger  suggested  the  name  "  toxalbumin." 

All  ptoraaius  contain  nitrogen  as  an  essential  part  of  their  basic 
character.  In  this  they  resemble  the  vegetable  alkaloids.  Some  of 
them  contain  oxygen,  while  others  do  not.  The  latter  correspond 
to  the  volatile  vegetable  alkaloids,  nicotin  and  coniin,  and  the  former 
correspond  to  the  fixed  alkaloids. 

The  kind  of  ptomain  formed  in  a  given  case  will  depend  upon  the 
individual  bacterium  engaged  in  its  production,  the  nature  of  the 
material  acted  upon,  and  the  conditions  under  which  the  bacterial 
growth  proceeds,  such  as  the  temperature,  the  amount  of  oxygen 
present,  and  the  duration  of  the  process.  For  instance,  Brieger 
found  that  although  the  Eberth  bacillus  grows  well  in  solutions  of 
pepton,  it  does  not  produce  any  ptomaifns ;  while  the  same  bacillus 
growing  in  beef  tea  elaborates  a  poisonous  alkaloid.  Fitz  found 
that  while  the  bacillus  butyricus  produces  by  its  action  on  carbohy- 
drates butyric  acid,  in  glycerin  it  forms  propylic  alcohol ;  and  Morin 
has  detected  amylic  alcohol  among  the  products  of  this  germ. 
Brown  has  shown  that  while  the  mycoderma  aceti  converts  ethylic 
alcohol  into  acetic  acid,  it  changes  propylic  alcohol  into  propionic 
acid,  and  is  without  effect  upon  methylic  alcohol,  primary  isobutylic 
alcohol,  and  amylic  alcohol.  Some  bacteria  will  not  multiply  below 
a  given  temperature ;  thus,  the  bacillus  butyricus  will  not  grow  be- 
low 24°.^ 

The  influence  of  the  presence  or  absence  of  certain  chemical  com- 
pounds upon  the  activity  of  unicellular  forms  of  life  has  long  been 
known  to  be  great.  Liebig  and  Knapp  observed  that  the  presence 
of  small  quantities  of  the  chlorids  of  sodium  and  potassium  intensifies 
the  action  of  yeast  in  the  decomposition  of  sugar,  and  Dubrumfaut 
made  quantitative  determinations  illustrating  the  effects  of  the  pres- 
ence of  other  salts.  He  found  that  0.5  gram  of  yeast  in  100  c.c.  of 
a  10  per  cent,  solution  of  sugar  decomposed  within  a  given  time  0.5 
gram  of  sugar,  while  in  another  flask  to  which  0.5  gram  of  sodium 
acid  sulphate  had  been  added,  the  same  amount  of  yeast  decomposed 
0.52  gram  of  sugar,  and  the  same  amount  of  potassium  sulphate  in  a 
second  flask  increased  the  activity  of  the  yeast  to  0.88  gram  of  sugar, 
and  in  a  third  flask  potassium  nitrate  raised  the  quantity  of  sugar  de- 
composed to  1  gram.  Pasteur  divided  bacteria  into  two  classes — the 
aerobic  and  the  anaerobic.     As  these  names  imply,  the  former  grow 

'  All  temperatures  given  in  this  work  are  centigrade,  unless  otherwise  specified 


BACTERIAL  PROTEIDS.  31 

and  thrive  in  the  presence  of  air,  while  the  latter  find  their  condi- 
tions of  life  improved  by  the  exclusion  of  air ;  therefore,  different 
products  will  be  formed  in  decomposing  matter  freely  exposed  to  the 
air,  and  in  that  which  is  buried  beneath  the  soil  or  from  which  the 
air  is  otherwise  largely  excluded.  Ignorance  of  this  fact  has  led  to 
some  serious  mistakes  in  toxicological  work,  as  we  shall  see  further  on. 

It  is  more  than  probable  that  most,  if  not  all,  ptomains  result 
from  the  cleavage  action  of  bacteria  on  the  constituents  of  the  me- 
dium in  which  they  grow,  and  it  follows  from  this  that  the  kind  of 
ptomain  found  in  a  given  case  will  depend  upon  the  stage  of  the  bac- 
terial disintegration  of  the  constituents  of  the  medium.  Regarded 
as  the  results  of  cleavage  action,  ptomains  are  transition  products  in 
the  processes  of  bacterial  decomposition.  They  are  temporary  forms 
through  which  matter  passes  while  it  is  being  changed,  by  the  ac- 
tivity of  bacterial  life,  from  the  organic  to  the  inorganic  state. 
Complex  organic  substances,  as  muscle  and  brain,  are  broken  up  into 
less  complex  molecules,  and  thus  the  process  of  chemical  division 
goes  on  until  the  simple  and  well-known  final  products,  carbonic  acid 
gas,  ammonia  and  water,  result ;  but  the  variety  of  combinations 
into  which  an  individual  atom  of  carbon  may  enter  during  this  long 
series  of  changes  is  almost  unlimited,  and  with  each  change  in  com- 
bination there  is  more  or  less  alteration  in  nature.  In  one  compound 
the  atom  of  carbon  may  exist  as  a  constituent  of  a  highly  poisonous 
substance,  while  the  next  combination  into  which  it  enters  may  be 
wholly  inert. 

It  was  formerly  supposed  that  putrefaction  was  simply  oxidation, 
but  the  researches  of  Pasteur  and  others  have  demonstrated  the  fact 
that  countless  myriads  of  microorganisms  are  engaged  constantly  in 
transforming  matter  from  the  organic  to  the  inorganic  form.  If  a  bit 
of  flesh  be  locked  up  so  that  these  little  workers  cannot  reach  it,  it  will 
remain  unchanged  indefinitely.  It  may  be  asked  if  any  of  the 
changes  occurring  during  putrefaction  are  to  be  regarded  as  purely 
chemical.  Without  doubt,  many  of  the  secondary  products  of  putre- 
faction arise  from  reactions  between  antecedent  and  more  complex 
substances,  or  by  the  action  of  oxygen,  water,  and  reducing  agents 
upon  primary  products.  Ptomains  formed  in  this  way  may  be  re- 
garded as  the  indirect  results  of  bacterial  life. 

While  some  of  the  basic  substances  formed  by  bacterial  growth 
are  intensely  poisonous,  it  is  not  probable  that  any  ptomain  can  be 
regarded  as  the  most  potent  product  of  one  of  the  pathogenic  bac- 
teria. It  follows,  therefore,  that  we  must  seek  for  the  specific  poisons 
of  the  infectious  diseases  among  other  chemical  compounds  produced 
by  bacterial  growth. 

Bacterial  Proteids. — Our  knowledge  concerning  these  substances 
still  remains  quite  imperfect  and  many  difficulties  arise  in  discussing 


32  CHEMICAL  PRODUCTS  OF  BACTERIA. 

them.  AVe  may  divide  the  bacterial  proteids  into  two  classes  :  (1) 
Those  which  constitute  an  integral  part  of  the  bacterial  cells ;  and 
(2)  those  which  have  not  been  assimilated  by  the  cells,  but  which 
have  been  formed  by  the  fermentative  or  cleavage  action  of  the  bac- 
teria on  the  proteid  bodies  in  which  they  are  growing.  This  classi- 
fication, however,  is  of  questionable  value.  We  allow  bacteria  to 
grow  for  a  number  of  days  in  a  nutrient  solution.  We  then  sep- 
arate the  soluble  constituents  from  the  formed  cells  by  filtration 
through  porous  tiles  ;  we  wash  the  latter  and  then  study  their 
proteid  contents  ;  but  a  considerable  proportion  of  the  contents  of  the 
living  cells  has  already  passed  into  solution,  and  the  bacterial 
detritus  left  on  the  filter  gives  no  exact  knowledge  of  the  con- 
stituents of  the  living  cells.  Moreover,  the  living  cells  absorb  and 
excrete,  and  we  are  not  yet  able  to  distinguish  with  certainty  be- 
tween those  substances  formed  within  the  cell  and  those  preexisting 
in  the  culture  medium.  The  filtrate  contains,  or  may  contain,  any 
one  or  more  of  the  following  proteid  bodies  :  (1)  Those  portions  of  the 
proteid  substance  which  were  used  in  the  preparation  of  the  nutri- 
ent solution  and  which  have  escaped  the  action  of  bacteria ;  (2) 
proteids  which  have  at  one  time  been  integral  parts  of  the  cells,  but 
which  have  passed  into  solution  on  the  death  and  dissolution  of  the 
bacteria ;  and,  (3)  proteids  which  have  been  formed  by  the  fermentative 
action  of  the  bacteria,  or  those  which  are  defined  as  constituting  the 
second  class,  as  given  above.  Attempts  are  now  being  made  (1901), 
by  growing  bacteria  on  solid  culture  media  extensively  to  distinguish 
with  certainty  the  proteid  constituents  of  the  cell  from  those  of  the 
culture  medium  ;  but  at  the  present  writing  this  work  has  not  pro- 
gressed sufficiently  for  us  to  make  any  positive  statements  concern- 
ing its  results. 

It  is  now  quite  certain  that  none  of  the  proteids  formed  by  the 
cleavage  action  of  bacteria  on  the  normal  constituents  of  culture 
media,  or  on  those  of  the  animal  body,  are  specific  factors  in  the 
production  of  disease.  It  is  true  that  many  bacteria  peptonize  pro- 
teids and  the  specific  poisons  of  certain  infectious  diseases  have  been 
sought  for  among  the  peptons ;  but  there  is  no  evidence  that  these 
are  more  poisonous  than  those  formed  by  the  gastric  juice.  The 
specific  bacterial  poisons  are  formed  by  synthetical  rather  than  by 
analytical  processes  and  they  are  constituents  of  the  protoplasm  en- 
closed in  the  cell  wall. 

The  Bacterial  Cellular  Proteids. — Nencki  was  the  first  to  at- 
tempt to  study  the  chemistry  of  the  bacterial  cell.  His  experiments 
were  made  with  putrefactive  bacteria,  which  were  obtained  by  de- 
cantation  of  liquid  cultures,  freed  from  fat  with  ether,  dissolved  in 
50  parts  of  a  potash  solution  of  0.5  per  cent.,  heated  for  some  hours 
at  100°  and  filtered.     The  filtrate  was  acidified  with  dilute  hydro- 


TOXINS.  33 

chloric  acid  and  precipitated  by  the  addition  of  rock  salt,  after  which 
the  precipitate  was  washed  with  saturated  salt  solution,  dried  at  100°, 
and  freed  from  salt  by  washing  with  water.  Nencki  designates  this 
substance  as  "mycoprotein,"  and  finds  that  it  has  the  formula 
^25-^42^6^3-  Freshly  prepared  mycoprotein  forms  amorphous  flakes, 
which  are  soluble  in  water,  alkalis  and  acids.  The  aqueous  solu- 
tion is  acid  in  reaction.  After  being  dried  at  100°  it  is  no  longer 
wholly  soluble  in  water.  Nencki  found  that  it  is  not  precipitated 
from  aqueous  solution  by  alcohol,  but  by  picric  acid,  tannic  acid  and 
mercuric  chlorid ;  that  it  does  not  give  the  xanthoproteic,  but  does 
give  the  Millon  and  the  biuret  reactions.  According  to  Schaffer,  it 
is  changed  by  acids  into  peptons,  and  on  being  fused  with  five  parts 
of  potash  it  breaks  up  into  ammonia,  amylamin,  phenol  (0.15  per  cent, 
of  its  weight),  valerianic  acid  (38  per  cent.),  leucin  and  traces  of 
indol  and  skatol.  A  proteid  obtained  from  the  yeast  plant  has  the 
formula  CjjHgjNgOg.  It  should  be  understood  that  these  formulse 
are  of  but  little  value  inasmuch  as  chemically  pure  bodies  were  not 
secured. 

The  pyogenetic  substance  obtained  from  the  pneumonia  bacillus 
of  Friedlander  was  found  by  Buchner  to  give  the  following  reac- 
tions :  It  is  soluble  in  water  and  the  concentrated  mineral  acids, 
very  soluble  in  dilute  alkalis,  from  which  it  is  precipitated  on 
the  addition  of  an  acid.  From  its  aqueous  solution  it  is  not  pre- 
cipitated by  heat,  nor  by  saturation  with  sodium  chlorid,  but  is  pre- 
cipitated by  magnesium  sulphate,  copper  sulphate,  platinum  chlorid, 
gold  chlorid,  lead  salts,  picric  acid,  tannic  acid,  and  absolute  alcohol. 
It  gives  the  xanthoproteic,  Millon  and  biuret  reactions. 

In  old  bouillon  cultures,  the  dead,  disintegrated  bodies  of  the 
bacteria  form  a  sediment.  It  is  not  at  all  probable  that  an  analysis 
of  this  sediment  represents  fairly  the  constituents  of  the  living  germs. 
During  the  disintegration  certain  constituents  of  the  cell  pass  into 
solution,  and  these  soluble  substances  are  probably  the  most  impor- 
tant parts  of  the  bacterial  cells,  so  far  as  they  are  concerned  in  the 
causation  of  disease.  Similar  processes  undoubtedly  occur  in  the 
body  of  an  animal  infected  with  a  pathogenic  organism  and  it  is 
generally  believed  that  these  soluble  substances  cause  the  symptoms 
of  the  disease  and  death. 

Toxins. — When  it  became  known  that  some  of  the  specific  patho- 
genic germs  elaborate  both  in  artificial  cultures  and  in  susceptible 
animals  poisonous  basic  substances  or  ptomains,  it  was  surmised  that 
the  symptoms  of  the  disease  induced  by  the  microorganisms  were 
due,  in  all  cases,  to  specific  basic  poisons,  and  chemists  labored  dili- 
gently to  isolate  from  cultures  of  each  germ  its  specific  toxic  products. 
These  labors  soon  led  to  the  recognition  of  the  fact  that  the  above- 
mentioned  surmise  had  been  hastily  drawn.  It  was  found  to  be  true 
3 


34  CHEMICAL  PRODUCTS  OF  BACTERIA. 

that  the  symptoms  of  each  and  every  individual  disease  investigated 
are  due  to  the  chemical  products  elaborated  by  the  activity  of  the 
germ,  but  these  chemical  products  are  not,  in  the  majority  of  these 
diseases,  basic  in  character,  and  consequently  they  cannot  be  classed 
among  the  ptomains.  Brieger  succeeded  in  isolating  from  pure 
cultures  of  the  tetanus  bacillus  as  many  as  four  ptomains,  but  the 
poisonous  effects  induced  by  these  substances  are  not  comparable  in 
violence  with  those  which  follow  injection  of  tetanus  cultures  from 
which  the  bacillus  has  been  removed  by  filtration.  The  fact  that  the 
filtered  culture  is  more  poisonous  than  any  or  all  of  its  basic  contents 
necessitates  the  conclusion  that  the  culture  contains  some  more  active 
constituent. 

What  is  the  nature  of  the  powerful  poisons  that  are  formed  in 
cultures  of  the  bacteria  of  tetanus,  diphtheria,  tuberculosis,  typhoid 
fever,  anthrax,  and  other  infectious  diseases,  and  which  are  also 
formed  in  the  bodies  of  animals  infected  with  these  microorganisms? 
At  present  no  positive  and  satisfactory  answer  can  be  made  to  this 
question.  At  one  time  Roux  and  Yersin  thought  that  it  might  be  a 
ferment,  and  Brieger  and  Fraenkel  advanced  the  belief  that  the  diph- 
theria poison  is  an  albuminous  body;  however,  there  are  weighty 
objections  to  each  of  these  theories,  and  up  to  the  present  time  all 
attempts  to  isolate  the  specific  toxins  have  proved  unsuccessful.  In 
1891  Freer  made  an  ultimate  analysis  of  a  toxin  obtained  by 
Vaughan  from  a  toxicogenic  germ  found  in  drinking  water,  and 
obtained  the  following  results :  Carbon,  48.46  per  cent. ;  hydrogen, 
7.69  per  cent.  ;  nitrogen,  13.44  per  cent.  ;  phosphorus,  0.69  per 
cent.  Sulphur  was  absent.  The  absence  of  sulphur  and  the  very 
small  per  cent,  of  phosphorus  were  supposed  at  that  time  to  indicate 
that  the  toxin  was  nearly  pure.  In  1893,  Brieger  and  Cohn  found 
that  the  tetanus  toxin,  in  the  purest  form  in  which  they  could  obtain 
it,  contained  no  phosphorus,  and  only  unweighable  traces  of  sulphur, 
and  they  attributed  the  presence  of  the  latter  element  to  contamina- 
tion with  ammonium  sulphate,  which  was  used  in  precipitation. 
Later  (1895),  Brieger  made  an  ultimate  analysis  of  the  tetanus  toxin, 
so  far  purified  that  0.000,000,05  gram  killed  mice,  with  the  following 
results  :  Carbon,  52.8  per  cent.  ;  hydrogen,  8.1  per  cent. ;  nitrogen, 
15.71  per  cent.  This  purified  toxin  is  not  precipitated  by  ammonium 
sulphate,  and  it  gives  the  biuret  reaction  so  imperfectly  that  Brieger 
felt  justified  in  saying  that  the  coloration  is  not  due  to  the  toxin,  but 
to  proteid  impurities. 

Uschinsky  has  made  an  important  contribution  to  our  knowledge 
of  the  toxins,  inasmuch  as  he  has  demonstrated  that  these  substances 
are  not  split  products,  formed  by  the  action  of  bacteria  on  proteids 
preexisting  in  the  culture  media,  but  are  synthetical  products,  and 
are  formed  when  the  germs  are  grown  in  culture  media  containing 
no  proteids.     He  succeeded  in  growing  a  number  of  pathogenic 


TOXINS.  35 

microorganisms,  including  those  of  cholera,  diphtheria,  tetanus  and 
typhoid  fever,  in  the  following  menstruum  : 


Water, 
Glycerin, 
Sodium  chlorid. 

1,000  parts, 
30-40     " 
5-7     " 

Calcium  chlorid. 

0.1  part. 

Magnesium  sulphate, 
Di-potassium  phosphate. 
Ammonium  lactate. 

0.2-0.4     " 
2-2.5  parts. 
6-7     " 

Sodium  asparginate, 

3.4     " 

From  these  cultures  he  obtained  toxins  which  were  not  less  virulent 
than  those  formed  in  ordinary  beef-tea,  and  thus  demonstrated  that 
their  production  is  due  to  synthetical  processes. 

Ehrlich,^  as  the  result  of  a  long  series  of  experiments  made  upon 
animals  with  toxins  and  antitoxins,  has  formulated  the  following 
conclusions  concerning  the  constitution  of  the  diphtheria  toxin  : 

1.  The  diphtheria  bacillus  produces  two  kinds  of  substances  :  (a) 
toxins,  (6)  toxons,  both  of  which  combine  with  anti-bodies.  In  fresh 
bouillon  cultures  toxins  and  toxons  exist  in  the  same  proportions. 

2.  The  toxins  (and  also  the  toxons)  are  not  simple  bodies,  but 
easily  split  up  into  other  substances  which  differ  from  one  another 
in  the  avidity  with  which  they  combine  with  antitoxins.  Among 
these  derivatives  there  are  found  prototoxins,  deuterotoxins  and 
tritotoxins,  which  are  here  mentioned  in  the  order  of  the  readiness 
with  which  they  combine  with  antitoxins,  the  tritotoxins  combining 
least  readily,  but  all  of  these  combine  with  antitoxins  more  readily 
than  do  the  toxons. 

3.  The  complication  does  not  stop  with  this  division,  but  it  is 
probable  that  every  toxin  consists  of  equal  parts  of  two  modifica- 
tions, which  behave  with  antitoxins  in  the  same  manner,  but  which 
differ  in  their  resistance  to  destructive  influences ;  probably  this 
difference  is  comparable  to  that  existing  between  dextro-rotatory  and 
levo-rotatory  forms  of  the  same  compound. 

4.  Of  these  two  modifications,  one,  which  may  be  designated  as 
the  a-modification,  readily  passes  into  a  toxoid.  Indeed,  this  trans- 
formation begins  and  frequently  reaches  completion  in  the  culture 
medium  in  the  incubator.  The  complete  conversion  of  the  a-modi- 
fication into  a  toxoid  leaves  the  substance  with  only  half  of  its 
original  toxicity,  and  it  may  therefore  be  called  a  hemitoxin. 

5.  The  second  modification,  which  is  designated  as  the  /?-modifica- 
tion,  varies  in  stability  in  the  different  kinds  of  poisons,  the  proto- 
toxins, the  deuterotoxins  and  the  tritotoxins.  The  /9-tritotoxin  is 
relatively  unstable  and  often  undergoes  decomposition  in  the  incu- 

1  Deutsche  med.  Wochenschrift,  September  22,  1898. 


36  CHEMICAL  PRODUCTS  OF  BACTERIA. 

bator.  The  /3-prototoxin  is  much  more  stable,  but  gradually  de- 
composes and  is  converted  into  a  toxoid  when  the  culture  is  kept 
for  several  months.  The  /3-modification  of  the  deuterotoxin  is  under 
certain  conditions  quite  stable.  These  facts  explain  the  observation, 
previously  reported,  that  old  cultures  of  diphtheria  gradually  de- 
crease in  toxicity  until  a  certain  minimum  degree  is  reached,  after 
which  there  is  no  further  decrease. 

6.  Avidity  for  combining  with  antitoxins  does  not  suffer  the 
least  change  on  the  conversion  of  a  toxin  into  a  toxoid  ;  for  instance, 
the  toxoid  of  prototoxin  combines  with  antitoxin  as  readily  as  does 
the  prototoxin  itself. 

7.  Those  varieties  of  poisons  which  combine  less  vigorously  with 
antitoxins  are  neutralized  by  the  same  reagent  less  promptly ;  this 
explains  the  observation  that  certain  poisons  of  the  tetanus  series 
(tetanolysin  and  tetanospasmin)  are  neutralized  promptly  by  anti- 
toxin only  in  concentrated  solution. 

8.  The  facts  observed  in  the  experiments  made  upon  animals  with 
toxins  and  antitoxins  are  best  explained  on  the  supposition  that  the 
toxic  molecule  contains  two  independent  groups  of  atoms,  one  of 
which  may  be  designated  as  the  haptophorous  and  the  other  as  the 
toxophorous  group.  It  is  by  the  action  of  the  haptophorous  group 
that  compounds  are  formed  with  antitoxins,  while  the  poisonous 
action  is  due  to  the  toxophorous  group.  The  toxons  also  possess 
these  two  groups  and  in  these  bodies  the  haptophorous  groups  are 
identical  with  those  of  the  toxins,  while  the  toxophorous  groups  are 
much  more  feeble  in  their  effects. 

9.  The  effects  of  the  haptophorous  and  the  toxophorous  groups  can 
in  certain  cases  be  experimentally  distinguished ;  for  instance,  Mor- 
genroth  found  that  when  frogs  were  kept  in  the  cold  and  successively 
injected  with  toxin  and  antitoxin,  these  animals  showed  no  evidences 
of  the  disease,  although  the  tetanus  poison  could  be  detected  in  their 
nerves.  On  the  other  hand,  when  frogs  were  treated  at  like  inter- 
vals with  toxin  and  antitoxin  and  kept  in  an  incubator,  they  suc- 
cumbed to  tetanus  even  when  all  of  the  poison  in  the  blood  was  com- 
bined with  antitoxin  and  an  excess  of  the  latter  was  present.  This 
indicates  that  the  haptophorous  group  combined  with  the  cells  in  the 
cold,  while  the  toxophorous  group  manifested  its  effects  only  when 
the  animal  was  kept  in  a  warm  place.  The  difference  in  time 
required  for  the  action  of  these  two  groups  possibly  explains  the  in- 
cubation period  which  is  observed  in  all  cases  of  poisoning  with  bac- 
terial toxins  ;  for  Donitz  has  shown  that  the  tetanus  poison  com- 
bines almost  instantaneously  with  the  nerve  cells. 

10.  The  toxophorous  group  is  more  complicated  in  structure  and 
therefore  less  stable  than  the  haptophorous.  The  relative  lability 
of  the  toxophorous  group  and  the  stability  of  the  haptophorous  group 
explain  the  quantitative  conversion  of  toxins  into  toxoids.     In  a 


TOXINS.  37 

structure  so  complicated  it  is  easy  to  conceive  of  an  asymmetrical 
atomic  arrangement  of  the  toxophorous  complex,  and  this  is  more 
easily  comprehensible  on  the  assumption  of  the  existence  of  the  two 
modifications  (a  and  ^)  in  the  same  amounts. 

11.  The  haptophorous  group,  which  under  ordinary  conditions  is 
quite  stable,  can  be  broken  down  by  the  action  of  strong  chemicals 
and  certain  physical  agents  (heat,  iodin,  etc.). 

12.  The  anti-bodies  combine  exclusively  with  the  haptophorous 
group  and  thus  absorb  the  toxic  molecule,  so  protecting  the  organism. 
In  this  way  the  poison  may  be  rendered  harmless  without  destruction 
of  the  toxophorous  complex. 

13.  It  follows  from  what  has  been  said  that  specific  antitoxins  can 
be  produced  not  only  with  toxins,  but  also  with  toxoids ;  indeed, 
highly  susceptible  animals  (mice  and  guinea-pigs),  can  be  immunized 
against  the  tetanus  toxin  safely  and  easily  by  means  of  toxoids.  It  is 
probable  that  in  this  case  the  degree  of  immunity  cannot  be  made 
so  great  as  is  desirable  in  the  preparation  of  a  curative  serum.  How- 
ever, it  is  possible  that  immunization  with  toxoids  can  be  used  di- 
rectly for  curative  purposes,  inasmuch  as  by  this  means  an  active 
immunity  may  be  brought  into  existence. 

14.  In  natural  immunity,  also  in  that  form  which  is  induced  by 
the  bacterium  itself  and  not  by  the  bacterial  poison,  the  toxons  prob- 
ably play  a  very  important  role ;  while  the  toxoids  do  not  exist  in 
these  cases  since  they  result  only  from  the  decomposition  of  toxins. 
It  is  also  probable  that  the  artificial  immunity  which  may  be  induced 
by  the  simultaneous  employment  of  an  antitoxic  serum  and  living 
bacteria  (as  in  rinderpest  and  swine  erysipelas),  and  which  is  quite 
lasting  and  is  produced  without  marked  disturbances  of  health,  is  in 
great  part  due  to  the  action  of  toxons. 

As  has  already  been  stated,  the  above-mentioned  theory  concern- 
ing the  constitution  of  toxins  is  founded  upon  experimental  studies 
of  the  action  of  toxins  and  antitoxins  in  the  animal  body,  and  does 
not  rest  upon  chemical  experimentation  ;  indeed,  Ehrlich  expresses 
the  opinion  that  many  decades  must  pass  before  we  shall  be  able  to 
isolate  the  toxin  chemically.  We  shall  have  opportunity  later  on  in 
our  studies  of  toxins,  antitoxins  and  immunity,  to  return  to  Ehrlich's 
theory.^ 

iSee  Chapter  Vn. 


CHAPTER  III. 

A  HISTOEICAL  SKETCH   OF   THE  BACTERIAL  POISONS. 

The  history  of  man's  experiments  with,  and  his  investigations  of, 
the  bacterial  poisons  quite  naturally  divides  itself  into  three  periods. 
During  the  first  period  man  was  ignorant  of  the  existence  of  bacteria 
and  of  the  role  that  they  play  in  processes  of  putrefaction,  as  well 
as  in  the  causation  of  disease,  and  in  his  experiments  he  did  not  al- 
ways adopt  measures  which  are  necessary  for  the  destruction  of  liv- 
ing microorganisms ;  consequently  the  results  obtained  were  in  some 
instances  due  wholly  to  infection,  in  others  to  bacterial  chemical 
poisons,  while  in  still  others  both  the  toxin  and  the  bacterium  prob- 
ably had  some  influence  in  causing  the  phenomena  which  he  ob- 
served. In  the  second  period  man  recognized  the  existence  of  bac- 
teria and  made  his  experiments  with  sterilized  products.  But  he 
had  not  at  that  time  secured  the  information  necessary  to  enable  him 
to  isolate  and  classify  the  microorganisms  ;  therefore,  the  toxins  with 
which  he  worked  resulted,  as  a  rule,  at  least,  from  the  growth  of 
mixed  cultures  of  germs,  the  characteristics  of  which  he  did  not 
know.  Indeed,  most  of  the  experiments  of  the  time  were  made 
with  sterilized  substances  obtained  from  putrefying  material.  The 
third  period  was  reached  when  the  experimenter  worked  with  pure 
cultures  and  for  the  first  time  was  able  to  proceed  scientifically  in 
his  investigations. 

It  must  have  been  known  to  primitive  man  that  the  eating  of 
putrid  flesh  was  liable  to  be  followed  by  more  or  less  harmful  effects, 
and  when  he  began  his  endeavors  to  preserve  his  food  for  future 
use,  instances  of  poisoning  from  putrefaction  must  have  multiplied. 
However,  the  distinguished  physiologist,  Albert  von  Haller,  seems 
to  have  been  the  first  to  make  any  scientific  experiments  concerning 
the  action  of  putrid  material  upon  animals.  He  injected  aqueous 
extracts  of  decomposing  flesh  into  the  veins  of  various  animals  and 
found  that  death  frequently  resulted.  Later  in  the  eighteenth  century, 
Morand  gave  an  account  of  the  symptoms  induced  in  man  by  eating 
putrid  meat.  In  the  early  part  of  the  nineteenth  century  (1 808—1814) 
Gaspard  carried  on  similar  experiments.  His  studies  were  made 
with  the  putrefied  flesh  of  both  carnivorous  and  herbivorous  animals. 
With  these  he  induced  marked  nervous  disturbances,  as  stiffness  of 
the  limbs,  opisthotonos  and  tetanus,  and  he  concluded  from  the 
symptoms  thus  induced  that  the  poisonous  effects  were  not  due  to 
carbonic  acid  gas  or  hydrogen  sulphid,  but  thought  it  possible  that 

38 


BACTERIAL  P0IS02iS.  39 

ammonia  might  have  part  in  their  production.  In  1820,  Kerner 
published  his  first  essay  on  poisonous  sausage,  and  this  was  followed 
by  a  second  in  1822.  At  that  time  he  thought  that  the  untoward 
symptoms  induced  by  eating  poisonous  sausage  were  due  to  a  fatty 
acid,  similar  to  the  sebacic  acid  of  Thenard,  and  which  originated 
during  putrefaction  ;  later,  he  modified  these  views  and  suggested 
that  the  poison  might  be  a  compound  consisting  of  sebacic  acid  and 
a  volatile  principle.  This  may  be  regarded  as  the  first  suggestion  as 
to  the  probability  of  a  poisonous  substance  with  basic  properties  in 
decomposing  matter.  In  1822  Dupr6  observed  a  peculiar  disease 
among  the  soldiers  under  his  care,  who,  during  the  warm  and  dry 
summer  of  that  year,  were  compelled  to  drink  foul  water ;  the  dis- 
ease thus  induced  was  probably  due  to  infection  rather  than  to  in- 
toxication. Later,  Magendie,  stimulated  by  the  investigations  of 
Gaspard  and  the  observations  of  Dupre,  made  many  experiments, 
in  which  dogs  and  other  animals  were  confined  over  vessels  contain- 
ing putrid  animal  matter  and  compelled  constantly  to  breathe  the 
emanations  therefrom.  The  effects  varied  markedly  with  the  species 
of  animal  and  the  nature  of  the  putrid  material,  but  in  some  in- 
stances symptoms  closely  resembling  those  of  typhoid  fever  in  man 
were  induced.  It  might  be  suggested  here  parenthetically  that  a 
repetition  of  the  experiments  of  Magendie,  with  such  precautions 
as  modern  methods  would  suggest,  would  not  be  without  value. 
Leuret  directed  his  attention  to  the  chemical  changes  produced  in 
blood  by  putrefaction,  but  accomplished  nothing  of  special  value. 
Dupuy  injected  putrid  material  into  the  jugular  vein  of  a  horse  and 
with  Trousseau  studied  alterations  produced  in  the  blood  by  these 
injections. 

During  the  third  decade  of  the  nineteenth  century  there  were  many 
investigators,  in  addition  to  those  mentioned  above,  who  endeavored 
to  ascertain  the  active  agent  in  poisonous  foods.  Dann,  Weiss,  Buch- 
ner,  Schumann,  Cadet  de  Gassicourt,  and  Orfila  studied  poisonous 
sausage,  but  made  no  advance  beyond  the  work  done  by  Kerner. 
Henneman,  Hiinnefeld,  Westrurab,  and  Sertiirner  (the  discoverer  ot 
morphin)  made  contributions  concerning  poisonous  cheese,  but  all 
reached  the  conclusion  that  the  caseic  acid  of  Kerner  is  the  poison- 
ous principle. 

In  1850,  Schmidt,  of  Dorpat,  studied  the  decomposition  products 
and  volatile  substances  found  in  cholera  stools,  and  two  years  later, 
Meyer,  of  Berlin,  injected  the  blood  and  stools  of  cholera  patients 
into  lower  animals.  In  1853,  Stich  made  an  important  contribution 
on  the  effects  of  acute  poisoning  with  putrid  material ;  he  ascertained 
that,  when  given  in  sufficient  quantity,  putrid  material  produced  an 
intestinal  catarrh,  with  choleraic  stools.  Nervous  symptoms,  tremb- 
ling, unsteady  gait,  and,  finally,  convulsions,  were  also  observed,  and 
careful  post-mortem  examinations  were  made,  but  no  important  or 


40         HISTORICAL  SKETCH  OF  THE  BACTERIAL  POISONS. 

characteristic  lesions  were  found.  He  concluded  that  the  putrid 
material  contained  a  ferment  which  induced  rapid  decomposition  of 
the  blood. 

The  distinguished  Danish  physiologist,  Panum,  was  the  first  to 
demonstrate  positively  the  chemical  character  of  the  poison  formed 
in  putrid  flesh,  inasmuch  as  he  showed  that  the  aqueous  extract  of 
such  material  retained  its  poisonous  properties  after  treatment  which 
insured  the  destruction  of  all  living  organisms.  Panum's  first  paper 
was  published  in  1856  and  his  conclusions  are  stated  as  follows  : 

1.  "The  putrid  poison  contained  in  the  decomposed  flesh  of  the 
dog,  and  which  is  obtained  by  extraction  with  distilled  water  and 
repeated  filtration,  is  not  volatile,  but  fixed.  It  does  not  pass  over 
on  distillation,  but  remains  in  the  retort. 

2.  "  The  putrid  poison  is  not  destroyed  by  boiling,  nor  by  evapora- 
tion. It  preserves  its  poisonous  properties  even  after  the  boiling  has 
been  continued  for  eleven  hours,  and  after  the  evaporation  has  been 
carried  to  complete  desiccation  at  100°. 

3.  "  The  putrid  poison  is  insoluble  in  absolute  alcohol,  but  is  solu- 
ble in  water,  and  is  contained  in  the  aqueous  extract  which  is  formed 
by  treating  with  distilled  water  the  putrid  material  which  has  been 
previously  dried  by  heat  and  washed  with  alcohol. 

4.  "  The  albuminoid  substances  which  frequently  are  found  in 
putrid  fluids  are  not  in  themselves  poisonous,  only  so  far  as  they 
contain  the  putrid  poison  fixed  and  condensed  upon  their  surfaces, 
from  which  it  can  be  removed  by  repeated  and  careful  washing. 

5.  "  The  intensity  of  the  putrid  poison  is  comparable  to  that  of 
the  venom  of  serpents,  of  curare,  and  of  certain  vegetable  alkaloids, 
inasmuch  as  0.012  of  a  gram  of  the  poison,  obtained  by  extracting 
with  distilled  water  putrid  material  which  had  been  previously  boiled 
for  a  long  time,  dried  at  100°,  and  submitted  to  the  action  of  ab- 
solute alcohol,  was  sufficient  to  almost  kill  a  small  dog.'' 

Panum  made  intravenous  injections  with  this  substance,  and  with 
ammonium  carbonate,  ammonium  butyrate,  ammonium  valerianate, 
tyrosin,  and  leucin,  and  found  that  the  symptoms  induced  by  the 
putrid  poison  dififered  from  those  caused  by  the  other  agents.  More- 
over, he  found  the  symptoms  induced  by  the  poison  to  differ  from 
those  of  typhoid  fever,  cholera,  pyemia,  anthrax,  and  sausage  poison- 
ing. He  was  in  doubt  whether  the  poison  acted  directly  upon  the 
nervous  system  or  as  a  ferment  upon  the  blood,  causing  decomposi- 
tion, the  products  of  which  might  affect  the  nervous  centers ;  but  he 
was  sure  that  his  new  substance  did  not  correspond  to  the  ordinary 
ferments,  inasmuch  as  it  was  not  decomposed  by  prolonged  boiling 
nor  by  treatment  with  absolute  alcohol.  Certainly,  Panum's  putrid 
poison  did  not  consist  of  living  organisms. 

The  symptoms  observed  by  Panum  varied  greatly  with  the 
quantity  of  the  poison  used  and  the  resistance  of  the  animal  experi- 


BACTERIAL  POISONS.  41 

merited  upon.  After  the  intravenous  injection  of  large  doses,  death 
followed  speedily.  In  these  cases,  there  were  violent  cramps  and 
involuntary  evacuations  of  the  urine  and  feces ;  the  respirations 
were  labored ;  pallor  was  marked,  sometimes  followed  by  cyanosis ; 
the  pulse  was  feeble  ;  the  pupils  were  widely  dilated,  and  the  eyes 
projected.  Autopsy  did  not  reveal  any  lesion,  save  that  the  blood 
was  dark  and  imperfectly  coagulated,  and  slightly  disseminated 
through  the  tissues.  Post-mortem  putrefaction  came  on  with  extra- 
ordinary rapidity. 

When  smaller  doses  or  more  vigorous  animals  were  used,  the 
symptoms  did  not  appear  before  from  a  quarter  of  an  hour  to  two 
hours,  sometimes  even  later,  and  were  not  violent,  generally  termi- 
nating in  recovery. 

In  addition  to  the  "  putrid  poison,"  Panum  obtained  a  narcotic 
substance,  the  two  being  separated  by  the  solubility  of  the  latter  in 
alcohol.  The  alcoholic  extract  was  evaporated  to  dryness,  the 
residue  dissolved  in  water  and  injected  into  the  jugular  vein  of  a 
dog.  The  animal  fell  into  a  deep  sleep,  which  remained  unbroken 
for  twenty-four  hours,  when  it  awoke  apparently  in  perfect  health. 
This  account  of  the  finding  of  a  narcotic  substance  in  putrefying 
flesh  is  rendered  more  interesting  by  the  later  researches  of  Bouchard, 
who  has  shown  that  normal  urine  contains  a  body  of  like  action, 
and  this  also  can  be  extracted  with  alcohol  from  the  residue  ob- 
tained by  evaporation. 

Weber,  in  1864,  and  Hemmer  and  Schwenninger,  in  1866,  con- 
firmed the  results  obtained  by  Panum,  and  Schwenninger  announced 
that  different  products  are  formed  in  the  various  stages  of  putrefac- 
tion and  that  these  are  unlike  in  their  effects  upon  animals.  In  1866, 
Bence  Jones  and  Dupre  obtained  from  the  liver  a  substance  which 
in  solutions  of  dilute  sulphuric  acid  gave  the  blue  fluorescence  ob- 
served in  similar  solutions  of  quinin  and  to  which  they  gave  the 
name  "animal  chinoidin."  Subsequently,  the  same  investigators 
found  this  substance  in  all  organs  and  tissues  of  the  body,  but  most 
abundantly  in  the  nerves.  These  observations  have  been  confirmed 
by  others,  and  solutions  showing  this  fluorescence  give  precipitates 
with  the  general  alkaloidal  reagents,  but  no  one  has  as  yet  succeeded 
in  isolating  the  basic  substance  supposed  to  be  present.  Indeed,  it 
is  not  positively  known  that  the  body  to  which  this  reaction  is  due 
is  a  bacterial  product,  although  this  is  the  most  likely  assumption. 
It  has  been  suggested  that  its  presence  might  be  due  to  the  growth 
of  certain  fluorescing  bacteria. 

In  1868,  Bergmann  and  Schmiedeberg  reported  that  they  had 
obtained  from  putrid  yeast  and  from  decomposed  blood  a  poisonous 
substance  in  the  form  of  a  sulphate  to  which  they  gave  the  name 
"  sepsin."  According  to  these  observers  the  sulphate  of  "  sepsin  " 
forms  in  needle-shaped  crystals,  and  small   doses  (0.01  gram),  dis- 


42         HISTORICAL  SKETCH  OF  THE  BACTERIAL  POISONS. 

solved  in  water  and  injected  intravenously  into  dogs,  cause  vomiting 
and  bloody  diarrhoea;  while  post-mortem  examination  shows  ecchy- 
moses  in  the  stomach  and  intestines.  It  was  then  believed  that  the 
"  putrid  poison  "  of  Panum  had  been  isolated,  and  that  it  was  iden- 
tical with  "sepsin."  However,  further  investigations  showed  that 
this  was  not  true,  and  it  is  by  no  means  certain  that  the  poisonous 
effects  obtained  by  Bergmann  and  Schmiedeberg  were  due  to  the 
crystals  seen  by  them  in  their  preparation,  inasmuch  as  these  crystals 
were  never  completely  isolated. 

Recently  Levy,  working  under  Schmiedeberg's  directions,  has 
made  further  study  of  putrid  yeast,  in  which  he  found  bacilli  re- 
sembling those  of  mouse  septicemia  and  the  proteus  vulgaris.  Dogs 
were  treated  with  intravenous  injections  of  liquefied  gelatin  cultures 
of  the  proteus,  and  symptoms  similar  to  those  formerly  attributed  to 
"  sepsin  "  followed.  The  cultures  were  precipitated  with  absolute 
alcohol,  and  the  resulting  albuminous  precipitate  caused  the  same 
symptoms  as  the  cultures  in  mice,  rabbits  and  dogs.  Levy  also  in- 
vestigated cases  of  meat  poisoning  due  to  infection  with  the  proteus. 
The  keeper  of  a  restaurant  and  some  of  his  guests  suffered  from  a 
most  violent  purging,  which  in  the  case  of  the  host  terminated 
fatally.  In  the  vomited  matter,  in  the  stools,  and  in  the  bottom  of 
the  ice-box  in  which  the  meat  was  kept,  the  proteus  was  found,  and 
cultures  of  it  produced  the  symptoms  of  sepsin  poisoning  in  animals. 
Levy  concludes  that  the  proteus  is  the  generator  of  sepsin.  If  this 
conclusion  be  correct,  it  follows  that  the  effects  observed  by  Berg- 
mann and  Schmiedeberg  were  not  due  to  the  crystals  obtained  by 
them,  but  to  other  substances  from  which  the  crystals  had  not  been 
separated. 

In  1869  Ziilzer  and  Sonnenschein  obtained  from  decomposing 
meat  extracts  a  nitrogenous  base  which,  both  in  its  chemical  reac- 
tions and  physiological  effects,  resembles  atropin  and  hyoscyamin. 
When  injected  under  the  skin  of  animals  this  substance  produces 
dilatation  of  the  pupils,  paralysis  of  the  muscles  of  the  intestines,  and 
acceleration  of  the  heart  beat ;  but  its  action  is  uncertain  and  incon- 
stant. A  similar  substance  has  been  obtained  from  the  bodies  of 
those  who  have  died  from  typhoid  fever,  and  it  is  possible  that  the 
belladonna-like  delirium  which  frequently  characterizes  the  later 
stage  of  this  disease  is  due  to  the  ante-mortem  generation  of  this  or  a 
similar  poison  within  the  body. 

During  the  eighth  decade  of  the  nineteenth  century  certain  toxi- 
cologists  became  interested  in  the  alkaloidal  reactions  obtained  some- 
times in  putrefying  tissue.  The  most  prominent  of  these  was  the 
Italian,  Selmi,  who  suggested  the  name  "  ptomain,"  and  whose  re- 
searches furnished  us  with  valuable  information,  and,  what  is  prob- 
ably of  more  importance,  gave  an  impetus  to  the  study  of  the 
chemistry  of  putrefactive  changes,  which  has  been  productive  of 


BACTERIAL  POISONS.  43 

much  good.  Selmi  showed  that  ptomams  could  be  obtained  (1) 
by  extracting  acidified  solutions  of  putrid  material  with  ether ;  (2) 
by  extracting  alkaline  solutions  with  ether  ;  (3)  by  extracting  alka- 
line solutions  with  chloroform ;  (4)  by  extracting  alkaline  solutions 
with  amylic  alcohol ;  and  (5),  that  there  yet  remain  in  the  solutions 
of  putrid  matter  ptomains  that  are  not  extracted  by  any  of  the  above- 
mentioned  reagents.  In  this  way  he  gave  some  idea  of  the  great 
number  of  alkaloidal  bodies  which  might  be  formed  among  the 
products  of  putrefaction,  and  the  promising  field  thus  discovered  and 
outlined  was  soon  occupied  by  a  busy  host  of  chemists.  In  the  sec- 
ond place,  he  demonstrated  the  fact  that  many  of  the  ptomams  give 
reactions  similar  to  those  observed  with  the  vegetable  alkaloids. 
This  led  the  toxicologist  into  investigations,  the  results  of  some  of 
which  we  will  ascertain  further  on.  Selmi  did  not  succeed  in  iso- 
lating any  putrefactive  alkaloid,  and  all  his  physiological  experiments 
and  chemical  reactions  were  made  with  extracts.  He  remained 
ignorant,  except  in  a  general  way,  of  the  composition  of  these  bodies. 
Nencki,  in  1876,  made  the  first  ultimate  analysis  and  determined  the 
first  formula  of  a  ptomai'n ;  this  was  an  isomer  of  collidin,  which 
will  be  described  later. 

Rorsch  and  Fassbender,  in  a  case  of  suspected  poisoning,  obtained 
a  liquid  which  could  be  extracted  from  acid  as  well  as  alkaline  solu- 
tions by  ether,  and  which  gave  all  the  general  alkaloidal  reactions ; 
but  they  were  unable  to  crystallize  and  isolate  it.  They  believed  that 
this  substance  was  derived  from  the  liver,  since  fresh  ox  liver  treated 
in  the  same  manner  gave  them  an  alkaloid  which  could  be  extracted 
from  ether  as  well  as  from  acid  or  alkaline  solution.  In  some  of  its 
reactions  this  substance  resembles  digitalin.  Gunning  found  a  sim- 
ilar body  in  liver  sausage  from  which  poisoning  had  occurred. 

Schwanert,  while  examining  the  decomposing  intestines,  liver  and 
spleen  of  a  child  that  had  died  suddenly,  perceived  a  peculiar  odor 
and  obtained  by  the  Stas-Otto  method  (ether  extract  from  alkaline 
solution)  small  quantities  of  a  base  which  was  distinguished  from 
nicotin  and  coniin  by  its  greater  volatility  and  its  odor.  He  sup- 
posed that  this  substance  was  produced  by  decomposition,  and  in 
order  to  ascertain  the  truth  of  this  supposition,  he  took  the  organs 
of  a  cadaver  that  had  lain  for  sixteen  days  at  a  temperature  of  30° 
and  was  well  decomposed.  These  were  treated  with  tartaric  acid  and 
alcohol,  and  the  acid  solution  was  first  extracted  with  ether  but 
yielded  no  result;  it  was  then  rendered  alkaline  and  extracted  with 
ether.  The  latter  extract  gave,  on  evaporation,  the  same  substance 
which  had  been  found  in  the  organs  of  the  child.  The  residue  was  a 
yellowish  oil,  having  an  odor  somewhat  similar  to  propylamin.  It 
was  repulsive,  but  not  bitter  to  the  taste,  and  alkaline  in  reaction. 
On  the  addition  of  hydrochloric  acid  it  crystallized  in  white  need- 
les, which  were  freely  soluble  in  water,  but  soluble  with  difficulty  in 


44        HISTORICAL  SKETCH  OF  THE  BACTERIAL  POISONS. 

alcohol.  On  the  addition  of  ammonium  hydrate  to  this  crystalline 
substance,  a  white  vapor  of  unpleasant  odor  was  given  off.  "When 
dissolved  in  sulphuric  acid  the  crystals  formed  a  solution  which  was 
at  first  colorless,  but  which  gradually  became  dirty  brownish-yellow, 
and  grayish-brown  on  the  application  of  heat.  On  being  warmed 
with  sodium  molybdate,  a  splendid  blue  color,  becoming  gradually 
gray,  was  produced.  Potassium  bichromate  and  sulphuric  acid  gave 
a  reddish-brown,  then  a  grass-green  color.  Nitric  acid  gave  a  yellow 
color.  A  tartaric  acid  solution  of  the  crystals  produced  on  the  addi- 
tion of  platinum  chlorid  a  dirty  yellow  precipitate  of  small  six-sided 
stars,  which  contain  31.55  per  cent,  of  platinum.  Gold  chlorid  gave 
a  pure  yellow,  amorphous  precipitate ;  mercuric  chlorid  yielded  white 
crystals  ;  potassio-mercuric-iodid  a  dirty  white  precipitate,  and  potas- 
sio-cadmic-iodid  yielded  no  result.  Tannic  acid  produced  only  a  tur- 
bidity. Sodium  phospho-molybdate  gave  a  yellow  flocculent  precip- 
itate which  became  blue  on  the  addition  of  ammonium  hydrate. 

Liebermann,  in  examining  the  somewhat  decomposed  stomach  and 
intestines  of  a  case  of  suspected  poisoning,  found  an  alkaloidal  body 
which  was  unlike  that  studied  by  the  chemist  mentioned  above,  inas- 
much as  it  was  not  volatile.  The  ether  extract  from  alkaline  solu- 
tion left,  on  evaporation,  a  brownish  resinous  mass  which  dissolved  in 
water  to  a  turbid  solution.  The  cloudiness  increased  on  heating. 
This  reaction  agrees  with  coniin,  but  the  odor  differed  from  that  of 
the  vegetable  alkaloid.  The  putrefactive  alkaloid  does  not  distil 
when  heated  on  the  oil-bath  to  200°,  while  coniin  distils  at  135®. 
The  former  is  with  certainty  distinguished  from  coniin  by  its  non- 
poisonous  properties.  This  substance  is  extracted  by  ether  from  acid 
as  well  as  from  alkaline  solutions ;  on  the  evaporation  of  the  ether  it 
appears  in  yellow  oily  drops,  which  are  soluble  in  alcohol. 

Selmi  obtained  from  both  putrefying  and  fresh  intestines  a  substance 
which  gave  the  general  alkaloidal  reactions  and  had  marked  reducing 
power.   When  warmed  with  sulphuric  acid  it  gave  a  violet  coloration. 

From  human  bodies  from  one  to  ten  months  after  death  Selmi  re- 
moved many  alkaline  bases.  From  an  ether  solution  of  one  of  these, 
one  basic  substance  was  removed  by  treatment  with  carbonic  acid  gas. 
Another  body  which  was  insoluble  in  ether,  but  readily  soluble  in 
amylic  alcohol,  was  found  to  be  a  violent  poison,  producing  in  rab- 
bits tetanus,  marked  dilatation  of  the  pupils,  choleraic  symptoms, 
and  death. 

Parts  of  a  human  body  preserved  in  alcohol  were  found  by  Selmi 
to  yield  an  easily  volatile,  phosphorus-containing  substance,  which 
was  soluble  in  ether  and  carbon  di-sulphid  and  gave  a  brown  pre- 
cipitate with  silver  nitrate.  It  was  not  the  phosphid  of  hydrogen. 
With  potassium  hydrate  it  gave  off  ammonia  and  yielded  a  substance 
having  an  intense  coniin  odor.  It  was  volatile  and  reduced  phospho- 
molybdic  acid.     A  similar  body  was  produced  by  the  slow  decom- 


BACTERIAL  POISONS.  45 

position  of  the  yelks  of  eggs.  Selrai  also  obtained  from  decomposing 
egg  albumin  a  body,  the  chlorid  of  which  formed  in  needles,  and 
possessed  a  curare-like  action  on  frogs.  From  one  arsenical  body, 
which  had  been  buried  for  fourteen  days,  he  obtained,  by  extracting 
a  solution  made  alkaline  with  baryta,  with  ether,  a  substance  which 
formed  in  needles  and  which  gave  crystalline  salts  with  acids.  With 
sulphuric  acid  it  gave  a  red  color ;  with  iodic  acid  and  sulphuric 
acid  it  liberated  free  iodin  and  gave  a  violet  coloration  ;  with  nitric 
acid  it  gave  a  beautiful  yellow  color  which  deepened  on  the  addition 
of  caustic  potash.  Platinum  chlorid  gave  no  precipitate  save  in 
highly  concentrated  solutions.  From  a  second  arsenical  body,  Selmi 
obtained  by  the  same  method  a  substance  which  gave  with  tannic 
acid  a  white  precipitate ;  with  iodin  in  hydriodic  acid  a  kermes- 
brown ;  with  gold  chlorid  a  yellow,  which  was  soon  reduced  ;  with 
mercuric  chlorid  a  white  ;  with  picric  acid  a  yellow,  which  gradually 
formed  in  crystalline  tablets.  This  substance  did  not  contain  any 
arsenic,  but  was  highly  poisonous.  From  the  stomach  of  a  hog 
which  had  been  preserved  in  a  solution  of  arsenious  acid  Selmi  sepa- 
rated an  arsenical  organic  base.  The  fluid  was  distilled  in  a  current 
of  hydrogen  ;  the  distillate,  which  was  found  to  be  strongly  alkaline, 
was  neutralized  with  hydrochloric  acid  and  evaporated  to  dryness, 
when  cross-shaped  crystals,  giving  an  odor  similar  to  that  of  trimethyl- 
amin,  were  obtained.  This  substance  was  found  by  Ciaccia  to  be 
highly  poisonous,  producing  strychnia-like  symptoms.  With  iodin 
in  hydriodic  acid  it  is  said  to  give  a  gray  crystalline  precipitate. 
From  the  liquid  which  remained  in  the  retort,  a  non-volatile  arsenical 
ptomain  was  extracted  with  ether.  An  aqueous  solution  of  this  gave 
with  tannic  acid  a  slowly  forming,  yellowish  precipitate  and  similarly 
colored  precipitates  with  iodin  in  hydriodic  acid,  platinum  chlorid, 
auric  chlorid,  mercuric  chlorid,  potassio-mercuric  iodid,  potassio- 
bismuthic  iodid,  picric  acid,  and  potassium  bichromate.  The  physi- 
ological action  of  this  substance,  as  demonstrated  on  frogs,  was  unlike 
that  of  the  arsines  and  consisted  of  torpor  and  paralysis. 

Moriggia  and  Battistini  experimented  with  substances  obtained 
from  decomposing  bodies,  upon  guinea-pigs  and  frogs,  but  did  not 
attempt  their  isolation  because  of  the  rapid  decomposition  which 
they  undergo  when  exposed  to  the  air  and  by  which  they  lose  their 
poisonous  properties.  These  alkaloids  were  found  to  be  easily 
soluble  in  amylic  alcohol,  less  soluble  in  ether. 

In  1871  Lombroso  showed  that  the  extract  from  mouldy  corn- 
meal  produced  tetanic  convulsions  in  animals.  (It  must  not  be  for- 
gotten that  similar  effects  may  be  due  to  the  cornutin  of  ergot.) 
This  threw  some  light  upon  the  cases  of  sporadic  illness  which  had 
long  been  known  to  occur  among  the  peasants  of  Lombardy,  who 
eat  fermented  and  mouldy  corn-meal.  In  1876  Brugnatelli  and 
Zenoni  obtained  by  the  Stas-Otto  method  from  this  mouldy  meal  an 


46        HISTORICAL  SKETCH   OF  THE  BACTERIAL  POISONS. 

alkaloidal  substance  which  was  white,  non-crystalline,  unstable  and 
insoluble  in  water,  but  readily  soluble  in  alcohol  and  ether.  With 
sulphuric  acid  and  bichromate  of  potassium  it  yields  a  color  reaction 
very  similar  to  that  of  strychnin. 

The  action  of  the  ether  extracts  from  decomposed  brain  resembled 
that  of  curare,  but  was  less  marked  and  more  transitory.  The 
beats  of  the  frog's  heart  were  decreased  in  number  and  strengthened 
in  force ;  the  nerves  and  muscles  lost  their  irritability,  and  the  ani- 
mal passed  into  a  condition  of  complete  torpor.  The  pupils  were 
dilated.  Guareschi  and  Mosso,  using  the  Stas-Otto  method,  obtained 
from  human  brains  which  had  been  allowed  to  decompose  at  a  tem- 
perature of  from  10°  to  15°  for  from  one  to  two  months,  both  vola- 
tile and  non-volatile  bases.  Among  the  former  only  ammonia  and 
trimethylamin  were  in  sufficient  quantity  for  identification.  With 
these,  however,  were  minute  traces  of  ptomams.  They  obtained 
non-volatile  bases  from  both  acid  and  alkaline  solutions.  From  the 
former  they  separated  a  substance  which  gave  precipitates  with 
gold  chlorid,  phosphotungstic  acid,  phosphomolybdic  acid,  Mayer's 
reagent,  palladium  chlorid,  picric  acid,  iodin  in  potassium  iodid,  and 
a  slight  one  with  tannic  acid.  This  substance  was  not  precipitated 
with  platinum  or  mercury. 

From  the  alkaline  extract  there  was  obtained  a  substance  which  in 
dilute  hydrochloric  acid  solutions  gave  with  gold  chlorid  a  heavy 
yellow  precipitate  with  reduction,  also  precipitates  with  phospho- 
molybdic acid,  platinum  chlorid,  Mayer's  reagent,  picric  acid,  phos- 
photungstic acid,  Mamie's  reagent,  iodin  in  potassium  iodid,  tannin, 
bichromate  of  potassium,  palladium  chlorid  and  mercuric  chlorid. 
It  reduced  ferric  salts.  From  decomposed  fibrin  the  same  investi- 
gators obtained  one  well-defined  ptomain.  Analyses  of  the  platinum 
compound  of  this  substance  gave  the  formula  Cj^Hj^N.  This  body 
will  be  discussed  in  a  future  chapter. 

From  fresh  brain  substance  they  separated  ammonia,  trimethyl- 
amin, and  an  undetermined  base.  These,  however,  are  not  to  be 
regarded  as  products  of  putrefaction,  but  as  resulting  from  the 
action  of  reagents  upon  the  brain  substance.  The  trimethylamin 
probably  arises  from  the  splitting  up  of  lecithin,  while  the  undeter- 
mined base  is  most  likely  cholin,  which  also  results  from  the  break- 
ing up  of  the  lecithin  molecule. 

They  also  show  that  when  DragendorfiP's  method  is  used  basic  sub- 
stances can  be  obtained  from  fresh  meat,  and  these  are  shown  to  be 
produced  by  the  action  of  sulphuric  acid  on  the  flesh. 

In  1885,  Vaughan  detected  in  poisonous  cheese  an  active  agent  to 
which  he  gave  the  name  "  tyrotoxicon,"  and  this  discovery  has  been 
confirmed  by  Newton, Wallace,  Schaeifer,  Stanton,  Firth,  Ladd,Wolff, 
Kimura,  Davis,  Kinnicut,  and  others  ;  however,  as  we  shall  see  later, 
this. is  not  the  substance  most  commonly  found  in  harmful  cheese. 


BACTERIAL  POISONS.  47 

From  1882  to  1888,  Brieger  succeeded  in  isolating  and  determin- 
ing the  composition  of  a  number  of  ptomains.  From  putrid  flesh 
he  obtained  neuridin,  C.Hj^Nj,  and  neurin,  C^HjgNO.  The  former 
is  inert,  while  the  latter  is  poisonous.  From  decomposing  fish  he 
separated  a  poisonous  base,  C2H^(NH2)2,  which  is  an  isomer  of  ethy- 
lidenediamin,  muscarin,  CgHj^NOg,  and  an  inert  base,  C.Hj^NOj, 
gadinin.  Rotten  cheese  yielded  neuridin  and  trimethylamin.  De- 
composed glue  gave  neuridin,  trimethylamin  and  a  muscarin-like 
base.  In  the  cadaver,  he  found  in  different  stages  of  decomposi- 
tion, cholin,  neuridin,  trimethylamin,  cadaverin,  C^Hj^Ng,  putrescin, 
C4HJ2N2,  and  saprin,  C.Hj^N2,  all  of  which  are  inert.  After  four- 
teen days  of  decomposition  he  found  a  poisonous  substance,  mydalein, 
and  from  a  cadaver  which  had  been  kept  at  from  —  9"  to  -|-  5°  for 
four  months,  he  obtained  mydin,  CgHj^NO,  the  poisonous  substance 
mydatoxin,  CgHj3N02,  also  the  poison  methyl-guanidin.  From 
poisonous  mussel  he  separated  mytilotoxin,  CgH,5N02. 

During  the  later  years  of  the  ninth  decade  of  the  nineteenth  cen- 
tury, chemists  began  to  study  the  products  of  bacterial  growth  in 
pure  cultures.  In  this  work,  Brieger,  following  a  method  devised 
by  himself,  succeeded  in  isolating  a  number  of  basic  substances, 
which  were  at  that  time  supposed  to  be  the  specific  toxins  of  certain 
diseases.  He  obtained  typhotoxin  from  cultures  of  the  typhoid  bac- 
illus, and  four  crystalline  bodies  in  growths  of  the  tetanus  organism. 
In  1888  Roux  and  Yersin  made  an  important  contribution  in  their 
classical  work  upon  the  diphtheria  toxin,  and  two  years  later  Brieger 
and  Friinkel  confirmed  these  discoveries  and  extended  this  line  of 
research.  Sewall,  Salmon  and  Smith  opened  up  a  fruitful  field  for 
investigation  by  showing  that  immunity  can  be  secured  with  chem- 
ical poisons,  the  first-mentioned  having  worked  with  snake  venom, 
while  the  others  used  sterilized  cultures.  A  few  years  later  this 
work  was  taken  up  and  greatly  advanced  by  Ehrlich  in  his  now 
classical  studies  on  immunity,  which  were  first  made  with  the  poison- 
ous principles  of  castor  bean  and  jequirity,  and  later  extended  to 
bacterial  products.  In  1887  Fodor  made  his  second  contribution  on 
the  germicidal  action  of  the  blood  in  vita.  This  work,  developed  by 
the  researches  of  Nuttall,  Buchner,  Metschnikoff,  Pfeiffer,  Ehrlich, 
Bordet  and  numerous  others,  has  greatly  advanced  our  knowledge  of 
the  manner  in  which  the  animal  organism  protects  itself  from  bac- 
terial invasion,  and  has  given  us  some  practical  tests,  as  the  Widal  re- 
action, which  are  useful  in  the  diagnosis  of  diseases.  In  1892,  blood 
serum  therapy,  which  had  been  attempted  before  by  Babes  and  Tiz- 
zoni,  was  brought  into  prominence  by  the  researches  of  Behring,  and 
confirmed  by  Roux  in  1894.  As  a  practical  result  of  this  line  of 
research  we  have  the  antitoxin  treatment  of  diphtheria,  probably  the 
most  brilliant  discovery  yet  made  within  the  domain  of  curative 
medicine.  All  of  these  subjects  will  be  dwelt  upon  in  detail  in  sub- 
sequent chapters. 


CHAPTER  IV. 

THE   BACTERIAL   POISONS   OF   SOME  OF   THE  INFECTIOUS 

DISEASES. 

We  will  now  give  our  attention  to  the  chemical  poisons  of  some 
of  the  infectious  diseases,  and  in  doing  this  we  will  illustrate,  sub- 
stantiate and  extend  the  statements  made  in  preceding  chapters. 

Anthrax. — The  definition  of  an  infectious  disease,  as  we  have  given 
it,  is  illustrated  by  the  facts  which  have  been  learned  concerning 
the  causation  of  anthrax,  which  has  probably  been  more  thoroughly 
studied  than  any  other  infectious  disease.  Kausch  taught  that  this 
disease  has  its  origin  in  paralysis  of  the  nerves  of  respiration,  but  as 
to  the  cause  of  this  paralysis  he  gave  no  information.  Delafond 
thought  that  anthrax  has  its  origin  in  the  influence  of  the  chemical 
composition  of  the  soil  affecting  the  food  of  animals  and  leading  to 
abnormal  nutrition.  The  investigations  of  Gerlach,  in  1845,  demon- 
strated the  contagious  nature  of  anthrax,  which  was  emphasized  by 
Husinger  in  1850  and  accepted  by  Virchow  in  1855.  However, 
as  early  as  1849  Pollender  found  numerous  rod-like  microorgan- 
isms in  the  blood  of  animals  sick  with  this  disease,  and  his  observa- 
tion was  confirmed  by  Brauell,  who  produced  the  disease  in  healthy 
animals  by  inoculations  with  matter  taken  from  an  anthrax  pustule. 
Attempts  were  made  to  ridicule  the  idea  that  this  organism  might 
be  the  cause  of  the  disease  but,  in  1863,  Davaine  showed  that  these 
rod-like  bodies  must  have  some  causal  relation  to  the  disease,  inas- 
much as  his  experiments  proved  that  inoculation  into  animals  of  the 
blood  of  those  sick  with  anthrax,  produced  the  disease  only  when 
taken  at  a  time  when  the  blood  contained  these  organisms.  He  also 
demonstrated  beyond  any  question  that  these  rod-like  bodies  are 
bacteria,  capable  of  growth  and  multiplication.  The  conclusions 
of  this  investigator  were  combated  by  many;  but  Pasteur,  Koch, 
Bollinger,  De  Bary,  and  others  studied  the  morphology  and  life 
history  of  these  organisms,  and  then  came  the  brilliant  results  of 
Pasteur  in  securing  protection  against  inoculated  anthrax  by  the  vac- 
cination of  healthy  animals  with  the  modified  germ  and  subsequent 
inoculation  with  the  virulent  form.  Then  the  question  arose.  How 
do  these  bacilli  produce  anthrax  ?  and  in  answer  to  this  question  the 
various  theories  which  we  have  mentioned  were  proposed. 

In  1877,  Pasteur  filtered  the  blood  of  animals  sick  with  anthrax, 
also  anthrax  cultures,  through  porcelain  and  injected  the  germ-free 
filtrate  into  animals  without  inducing  the  disease,  and    concluded 

48 


ANTHRAX.  49 

that  this  bacillus  does  not  produce  any  soluble  poison.  The  first 
successful  attempt  to  study  the  chemical  poison  of  anthrax  was  made 
by  Hoifa,  who  obtained  from  pure  cultures  of  the  bacillus  small 
quantities  of  a  ptomain  which,  when  injected  under  the  skin  of 
animals,  produces  the  symptoms  of  the  disease  and  death.  This 
substance  causes  at  first  increased  respiration  and  action  of  the  heart, 
then  the  respirations  become  deep,  slow  and  irregular ;  the  temper- 
ature falls  below  the  normal,  the  pupils  are  dilated,  and  a  bloody 
diarrhoea  sets  it.  On  section  the  heart  is  found  contracted,  the 
blood  dark,  and  ecchymoses  are  observed  on  the  pericardium  and 
peritoneum.  Hoffa  named  this  poison  anthracin,  and  later  he  re- 
ported that  he  had  succeeded  in  isolating  it  from  the  bodies  of  ani- 
mals dead  from  anthrax.  It  must  be  admitted  that  Hoffa's  claims 
are  not  altogether  satisfactory,  and  that  they  lack  confirmation. 
Moreover,  the  small  amount  of  the  basic  substance  which  he  ob- 
tained renders  it  highly  probable  that  in  the  case  of  a  germ  so  viru- 
lent as  that  of  anthrax,  there  must  be  other  chemical  poisons  pro- 
duced. In  1889  Hankin,  by  growing  the  anthrax  bacillus  for  some 
days  in  a  nutritive  solution  consisting  of  Liebig's  meat  extract  to 
which  fibrin  had  been  added,  filtering  and  treating  the  filtrate  with 
ammonium  sulphate,  obtained  an  albumose  which,  while  not  directly 
poisonous  to  animals,  when  injected  simultaneously  with  an  inocula- 
tion of  the  anthrax  bacillus,  causes  more  speedy  death  than  when 
the  bacillus  only  is  used.  From  these  observations  Hankin  con- 
cluded that  the  albumose  destroys  the  natural  resistance  of  the  ani- 
mal to  the  disease,  after  which  the  bacillus  is  able  to  continue  the 
elaboration  of  the  poison  in  the  animal  body. 

Martin,  by  growing  the  anthrax  bacillus  for  from  ten  to  fifteen 
days  in  an  alkaline  albuminate  from  blood  serum,  and  then  by  filtra- 
tion through  porcelain,  obtains  the  following  products  : 

1.  Protoalbumose  and  deuteroalbumose,  and  a  trace  of  pepton, 
all  of  which  react  chemically  like  similar  substances  prepared  by 
peptic  digestion. 

2.  An  alkaloid. 

3.  Small  quantities  of  leucin  and  tyrosin. 

The  most  characteristic  property  of  the  albumoses  is  that  their 
solutions  are  strongly  alkaline,  and  the  alkalinity  is  not  removed  by 
ether,  or  by  dialysis. 

The  alkaloid  is  soluble  in  water,  alcohol  and  amylic  alcohol ;  and 
is  insoluble  in  chloroform,  ether  and  benzol.  Its  solutions  are 
strongly  alkaline  and  the  alkaloid  forms  crystalline  salts  with  acids. 
It  is  precipitated  by  the  general  alkaloidal  reagents,  with  the  excep- 
tion of  potassio-mercuric  iodid.  It  is  somewhat  volatile  and  loses  its 
poisonous  properties  on  exposure  to  the  air. 

The  mixed  albumoses  are  poisonous  only  when  considerable  doses 
are  taken,  0.3  gram  being  required  to  kill  a  mouse  of  22  grams 
4 


50  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

weight  when  injected  subcutaneously.  Smaller  doses  cause  a  local 
oedema  and  a  somnolent  condition,  from  which  the  animal  recovers. 
The  larger  doses  produce  a  more  extensive  oedema,  and  the  somno- 
lence deepens  to  coma,  terminating  in  death.  In  some  cases  the 
spleen  is  enlarged.  The  absence  of  germs  was  determined  by  plate 
cul  ures.  The  alkaloid  causes  similar  symptoms.  It  is,  however, 
more  poisonous,  and  acts  more  rapidly  than  the  albumoses.  The 
animal  is  affected  immediately  after  the  injection,  and  the  gradually 
increasing  coma  terminates  in  death.  The  alkaloid  also  causes 
oedema  and  in  many  cases  thrombosis  of  the  small  veins.  Extrav- 
asations into  the  peritoneal  cavity  are  occasionally  seen,  and  the 
spleen  is  ordinarily  enlarged  and  filled  with  blood.  The  fatal  dose 
for  a  mouse  is  from  0.1  to  0.15  gram,  death  resulting  within  three 
hours.  This  alkaloid  does  not  appear  to  be  identical  in  its  action 
with  the  anthracin  of  Hoffa.  Martin  also  succeeded  in  obtaining 
like  substances  from  the  bodies  of  infected  animals.  He  regards  the 
albumose  as  an  antecedent  of  the  ptomain.  It  is  probable  that  the 
albumoses  obtained  by  Han  kin  and  Martin  are  identical.  Balp  and 
Carbone  ^  succeeded  in  isolating  from  the  oedematous  tissue  of  an 
animal  dead  with  anthrax  an  albuminous  body  of  slight  toxicity,  and 
a  similar  result  was  obtained  by  Landi.  ^  Maltzew  ^  found  that  when 
from  0.2  to  7  c.c.  of  a  filtered  bouillon  culture  of  the  anthrax  germ 
was  injected  subcutaneously  in  rabbits,  it  induced  no  symptoms,  but 
if  the  same  animals  were  inoculated  with  anthrax  within  from  ten  to 
eighteen  days  they  died  much  more  speedily  than  control  animals. 
It  will  thus  be  seen  that  Hankin's  results  have  been  several  times 
confirmed.  However,  it  should  be  stated  that  Petermann  *  claims  to 
have  repeated  Hankin's  experiments  and  obtained  an  albumose,  the 
only  effect  of  which  was  to  elevate  the  temperature  from  one  to  two 
degrees,  and  Klemperer^  obtained  from  boiled  anthrax  cultures  an 
albuminous  substance  which  caused  elevation  of  temperature.  Han- 
kin  and  Wesbrook  ^  repeated  the  former's  experiments,  using  a  so- 
lution of  pure  pepton  as  a  culture  medium.  After  some  weeks,  the 
culture  was  filtered,  treated  with  ammonium  sulphate,  centrifuged, 
dialyzed,  and  precipitated  with  alcohol.  In  this  way  an  albumose  of 
slight  toxicity  was  obtained. 

Brieger  and  Frankel  endeavored  to  prepare  a  toxalbumin  from  the 
organs  of  animals  dead  with  anthrax.  The  finely  divided  tissue  was 
thoroughly  rubbed  up  with  water,  allowed  to  stand  for  twelve  hours 
in  an  ice-box  and  filtered  through  porcelain.  The  filtrate  was  evapo- 
rated in  vacuo  at  30°  to  one-third  its  volume,  and,  after  being  acidi- 

'  Giornale  della  R.  accademia  di  medicina  di  Torino,  1891. 

^RivisUi  gev£rale  italiana  di  clinica  medica,  1891. 

'  Russkaia  Medicina,  1891. 

*  Annaies  de  I'  InHliiut  Pasteur,  1 892. 

^ 2!eitschrifi /.  klin.  Median.,  1892. 

^Annaies  de  H Inatitut  Pasteur,  1892. 


ANTHRAX.  51 

fied  with  a  few  drops  of  acetic  acid,  was  treated  with  ten  times  its 
volume  of  absolute  alcohol.  This  mixture  was  allowed  to  stand  for 
twelve  hours  longer  in  an  ice-box,  after  which  the  precipitate  was 
collected  on  a  filter,  dissolved  in  a  small  volume  of  water,  refiltered, 
and  reprecipitated  with  alcohol,  this  being  repeated  until  a  perfectly 
clear  aqueous  solution  was  obtained.  The  albumose  was  further 
purified  by  dialysis,  and  as  thus  obtained,  it  was  found  to  be  freely 
soluble  in  water  and  to  give  the  ordinary  reactions  for  albumin. 
The  toxicity  of  this  albumose  is  much  greater  than  that  of  similar 
substances  obtained  by  others. 

Marmier  grew  anthrax  germs  in  the  following  medium  : 

Water,  1,000  grams. 
Pepton,  40       " 

Sodium  chlorid,  15       " 

Sodium  phosphate,  .5    " 

Potassium  phosphate,  .2    " 

Glycerin,  10      " 

The  pepton  used  was  obtained  from  the  commercial  preparation  by 
precipitation  of  the  other  proteids  with  ammonium  sulphate  and  by 
removing  the  salt  by  dialysis.  In  this  menstruum  the  anthrax 
bacillus,  especially  the  sporeless  variety,  grew  abundantly.  The 
toxin  was  obtained  from  the  culture  medium  by  precipitation  with 
ammonium  sulphate.  When  dried  it  is  soluble  in  water  and  in  a 
one  per  cent,  solution  of  phenol ;  insoluble  in  chloroform,  ether,  and 
absolute  alcohol.  It  is  said  not  to  give  any  of  the  reactions  of  albu- 
minoids, propeptons,  peptons,  or  alkaloids,  but  since  there  is  no  men- 
tion of  the  reactions  tested,  and  since  precipitation  with  ammonium 
sulphate  is  a  pepton  and  propepton  reaction,  this  statement  must  be 
considered  as  somewhat  indefinite  and  possibly  misleading.  There 
is  no  proof  that  the  toxin  obtained  was  pure ;  in  fact,  the  report 
makes  the  reader  certain  that  the  final  product  was  by  no  means 
chemically  pure.  The  author  was  surprised  in  studying  the  poison- 
ous effects  of  this  toxin  to  find  that  while  twenty  milligrammes  killed 
some  rabbits  readily,  others  resisted  seventy  milligrammes  ;  and  it 
does  not  seem  to  have  occurred  to  him  that  this  might  be  due  to  the 
fact  that  the  most  of  his  product  was  inert,  and  that  these  differences 
in  effects  were  due  to  the  unequal  distribution  of  the  active  agent. 
However,  we  will  give  the  author's  conclusions  as  he  states  them : 

1 .  A  specific  toxin  may  be  extracted  from  glycerin-pepton  cultures 
of  the  anthrax  bacillus. 

2.  This  toxin  does  not  give  the  reactions  of  albuminoid  substances. 
It  does  not  change  starch,  sugar  or  glycogen. 

3.  The    animals  (chickens,  frogs,  fish)  that    are  immune  to  the 
anthrax  bacillus,  are  also  indifferent  to  the  toxin.     Similar  results 


62  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

were  observed  in   rabbits  artificially  immunized  with   attenuated 
cultures. 

4.  This  toxin  is  attenuated,  but  not  destroyed,  by  boiling  at  110", 
thus  differing  from  the  venom  of  serpents,  the  toxins  of  diphtheria 
and  tetanus,  and  the  enzymes. 

5.  On  the  contrary,  like  the  other  bacterial  toxins,  it  loses  its 
effect  on  animals  after  being  brought  in  contact  with  the  alkaline 
hypochlorites.  Prolonged  insolation  in  the  presence  of  air  leads  to 
the  same  result. 

6.  By  employing  carefully  graduated  doses  of  this  toxin  it  is 
possible  to  immunize  animals  to  the  anthrax  bacillus  in  the  same 
way  as  other  specific  toxins  give  immunity  to  the  corresponding 
disease. 

7.  Anthrax  cultures  in  other  fluids,  such  as  blood  serum,  and 
bouillon  from  the  flesh  of  the  horse,  ox,  or  calf,  do  not  contain  the 
toxin  in  appreciable  quantities. 

8.  On  the  contrary,  one  may  extract  a  toxin  from  anthrax  cul- 
tures on  nutritive  gelatin  by  macerating  the  microorganisms  in 
alcohol  water  (20  per  cent,  alcohol). 

The  toxin  is  contained  within  the  bacterial  cells,  and  in  order  to 
obtain  it  in  the  culture  there  must  be  opportunity  for  it  to  diffuse 
from  the  cells. 

Heim  and  Geyger  ^  grew  anthrax  bacilli  in  eggs,  after  the  manner 
of  Hueppe,  extracted  with  alcohol,  precipitated  with  mercuric  chlorid, 
filtered,  treated  the  filtrate  mth  platinum  chlorid,  decomposed  this 
precipitate  with  hydrogen  sulphid,  rendered  the  filtrate  alkaline  with 
potassium  hydrate,  and  extracted  one  portion  with  ether,  and  another 
with  benzol.  The  benzol  residue,  when  taken  up  with  water,  killed 
mice  ;  the  ether  residue  was  less  toxic. 

Ivanow^  has  shown  that  certain  volatile  fatty  acids,  formic,  acetic 
and  caproic,  may  be  formed  in  anthrax  cultures ;  but  there  is  no 
evidence  that  the  production  of  these  substances  has  anything  to  do 
with  the  pathology  of  the  disease. 

Petri  and  Maassen  ^  have  detected  hydrogen  sulphid  in  anthrax 
cultures ;  but  spectroscopic  examination  of  anthrax  blood  failed  to 
show  the  presence  of  this  substance,  and  there  is  no  evidence  that 
this  gas  has  anything  to  do  with  the  production  of  the  disease. 

Fermi  has  shown  the  presence  of  peptonizing  and  diastatic  fer- 
ments in  anthrax  cultures ;  while  Maumus  has  detected  a  sugar- 
forming  agent,  and  Roger  reports  the  presence  of  a  rennet.  Klein  * 
removed  the  germs  from  agar  cultures  of  forty-eight  hours'  growth, 
placed  the  same  in  5  c.c,  of  bouillon,  and  after  the  tube  had  been 

^  Lehrbuch  der  Bakt.  TJntersuchung,  1894. 
2  Annales  de  I'  Institut  Pasteur,  1892. 
'^  Arbeiten  aus  dem  k.  Oesundheitsamte,  8,  318. 
*  Centralblatt  f.  Bakleriologie,  15,  598. 


ANTHRAX.  53 

held  for  five  minutes  in  boiling  water,  injected  two-thirds  of  its  con- 
tents into  the  peritoneal  cavity  of  a  guinea-pig  without  results. 
From  this  he  concludes  that  the  anthrax  bacillus  does  not  form  any 
intracellular  poison. 

Bianchi-Mariotti  ^  has  studied  the  effects  of  the  soluble  constitu- 
ents of  anthrax  cultures  on  the  isotonia  of  the  blood.  He  found 
that  the  intravenous  injection  of  cultures  filtered  through  porcelain, 
in  small  or  medium  doses,  increased  the  isotonic  properties  of  the 
blood  of  the  rabbit,  but  in  larger  doses  diminished  the  same;  fur- 
thermore, he  showed  that  the  amount  of  hemoglobin  is  decreased 
after  the  injection,  in  direct  proportion  to  the  quantity  of  the  cul- 
ture used. 

Conradi  ^  has  endeavored  to  solve  the  question  of  the  existence  of 
an  anthrax  toxin  by  the  following  methods. 

1.  The  exudates  which  form  in  the  peritoneal  cavities  of  guinea- 
pigs  inoculated  with  anthrax,  were  filtered  through  both  Kitasato 
and  Chamberland  filters  and  injected  into  susceptible  animals  with- 
out effect.  The  amount  of  filtered  extract  injected  into  rabbits 
varied  from  10  to  20  c.c. 

2.  The  livers  and  spleens  of  guinea-pigs,  which  had  succumbed 
to  anthrax,  were  immediately  after  death  rubbed  up  with  sterilized 
sand  in  a  sterilized  mortar,  diluted  with  physiological  salt  solution, 
filtered  through  porcelain  and  injected  into  white  rats,  guinea-pigs 
and  rabbits  without  effect. 

3.  Collodion  sacs  filled  with  virulent  anthrax  cultures,  were 
placed  in  the  abdominal  cavities  of  susceptible  animals,  where  they 
remained  without  apparent  detriment  to  health.  This  was  a  repe- 
tition of  similar  experiments  made  previously  by  Sanarelli  and 
Pekelharing. 

4.  The  anthrax  exudates  in  quantities  of  from  5  to  6  c.c.  were 
placed  in  test-tubes,  |  c.c.  of  toluol  added,  the  tube  closed  with  steril- 
ized cork,  thoroughly  shaken,  and  then  allowed  to  stand  for  ten  days 
in  the  dark  at  room  temperature.  At  the  expiration  of  this  time  the 
contents  of  many  tubes  were  placed  in  a  separator  and  the  toluol  re- 
moved. The  exudate  in  which  the  germ  had  thus  been  destroyed 
by  toluol  was  injected  into  susceptible  animals  without  effect. 

5.  Having  shown  that  asporogenic  cultures  are  deprived  of  vi- 
tality by  exposure  for  110  hours  to  —  16°,  such  cultures,  after  being 
thus  sterilized  and  being  kept  for  some  time  in  the  incubator  to 
prove  their  sterility,  were  injected  into  susceptible  animals  without 
effect. 

6.  Thinking  it  possible  that  a  toxin  might  be  extracted  under 
pressure,  after  the  method  used  by  Buchner  in  obtaining  a  ferment 
from  yeast,  cultures  were  exposed  to  hydraulic  pressure  of  500  at- 

^  Wiener  med.  Presse,  1894. 
'Zeitschriftf.  Hygiene,  31,  237. 


54  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

mospheres.  The  resulting  fluid  was  filtered  through  porcelain  and 
injected  into  animals  without  effect. 

7.  The  experiment  of  Brieger  and  Frankel,  in  which  they  pre- 
pared their  anthrax  toxalbumin,  was  repeated  with  a  negative  result. 

From  these  experiments  Conradi  reaches  the  following  conclusion  : 
"  By  no  method  known  at  present  can  it  be  shown  that  the  anthrax 
bacillus  forms  either  an  extra-cellular  or  an  intra-cellular  poison  in 
the  animal  body ;  indeed,  these  experiments  increase  the  probability 
that  the  anthrax  bacillus  does  not  form  any  poisonous  substance. 
Therefore  the  solution  of  the  manner  in  which  anthrax  infection  re- 
sults remains  unknown.  Whether  improved  chemical  methods  will 
lead  to  the  detection  of  a  poison  or  not  cannot  be  predicted,  but  for 
the  present  the  anthrax  bacillus  must  be  regarded  as  a  purely  infec- 
tious microorganism."  If  this  be  true,  the  mechanical  interference 
theory  is  the  best  that  can  at  present  be  offered  so  far  as  anthrax  is 
concerned.^ 

Asiatic  Cholera. — Bitter  has  shown  that  the  comma  bacillus  pro- 
duces in  meat  pepton  cultures  a  peptonizing  ferment,  which  remains 
active  after  the  organism  has  been  destroyed.  Like  similar  chemical 
ferments,  it  converts  an  indefinite  amount  of  coagulated  albumin  into 
pepton.  It  is  more  active  in  alkaline  than  in  acid  solutions,  thus 
resembling  pancreatin  more  than  pepsin.  The  resemblance  to  pan- 
creatin  is  further  shown  by  increased  activity  in  the  presence  of  cer- 
tain chemicals,  such  as  sodium  carbonate  and  salicylate.  Bitter  also 
demonstrated  that  this  microorganism  produces  a  diastatic  ferment, 
inasmuch  as  he  found  that  it  develops  an  acid  in  nutrient  solutions 
containing  starch  paste  ;  however,  all  attempts  to  isolate  this  ferment 
were  unsuccessful.  A  temperature  of  60°  destroys  or  markedly  de- 
creases the  activity  of  ptyalin,  and  this  is  also  true  of  the  diastatic 
ferment  produced  by  the  comma  bacillus. 

Fermi  isolated  the  peptonizing  ferment  of  the  cholera  germ  in  the 
following  manner  :  65  per  cent,  alcohol  added  to  gelatin  which  has 
been  liquefied  by  the  bacillus,  precipitates  the  proteid,  but  not  the 
ferment ;  after  twenty-four  hours  the  precipitate  is  removed  by  fil- 
tration, and  the  ferment  precipitated  from  the  filtrate  by  the  addition 
of  absolute  alcohol.  After  being  collected  on  a  filter  and  dried,  this 
ferment  may  be  dissolved  in  an  aqueous  solution  of  thymol  and  its 
peptonizing  properties  demonstrated  on  gelatin  tubes. 

Rietsch  believes  that  the  destructive  changes  observed  in  the  in- 
testines in  cholera  are  due  to  the  action  of  the  peptonizing  ferment. 

Cantani  injected  sterilized  cultures  of  the  comma  bacillus  into  the 
peritoneal  cavities  of  small  dogs  and  observed  after  from  one-quarter 
to  one-half  hour  the  following  symptoms  :  great  weakness,  tremor  of 
the  muscles,  drooping  of  the  head,  prostration,  convulsive  contractions 

'  See  page  20. 


ASIATIC  CHOLERA.  55 

of  the  posterior  extremities,  repeated  vomiting,  and  cold  head  and 
extremities.  After  two  hours  these  symptoms  began  to  abate,  and 
after  twenty-four  hours  recovery  seemed  complete.  Control  experi- 
ments with  like  amounts  of  uninfected  beef  tea  gave  negative  results. 
The  cultures  used  were  three  days  old  when  sterilized.  Older  cul- 
tures seemed  less  poisonous,  and  a  high  or  prolonged  heat  in  sterili- 
zation decreased  the  toxicity  of  the  fluid  ;  therefore,  Cantani  concludes 
that  the  poisonous  principle  is  volatile,  but  the  effect  of  high  or  pro- 
longed heat  in  diminishing  the  toxicity  was  more  probably  due  to 
destructive  action  on  the  toxin.  The  same  observer  found  that  the 
blood  of  those  sick  with  cholera  is  acid ;  this  has  been  confirmed  by 
Strauss  on  examining  the  blood  directly  after  death,  and  Ahrend 
found  lactic  acid  in  the  strongly  acid  urine  of  a  cholera  patient. 

Nicati  and  Rietsch  killed  dogs  by  injecting  intravenously  cultures 
from  which  all  the  germs  had  been  removed  by  filtration,  and  they 
also  obtained  from  old  bouillon-pepton  cultures  a  poisonous  base. 
Van  Ermengem  also  found  that  cultures  after  filtration  through  a 
Chamberland  filter  are  poisonous. 

Klebs  studied  the  cholera  toxin  in  the  following  manner  :  Cul- 
tures in  fish  preparations  were  acidified,  filtered ;  the  filtrate  evapo- 
rated on  the  water-bath ;  the  residue  taken  up  with  alcohol  and  pre- 
cipitated with  platinum  chlorid.  The  platinum  was  removed  with 
hydrogen  sulphid  and  the  crystalline  residue  obtained  on  evaporation 
was  dissolved  in  water  and  injected  into  rabbits  intravenously. 
Muscular  contractions  were  induced,  and  death  followed  in  one  ani- 
mal, which,  in  addition  to  the  above  treatment,  received  an  injection 
of  a  non-sterilized  culture.  In  the  latter  case  the  epithelium  of  the 
uriniferous  tubules  was  found  to  be  extensively  calcified.  Klebs 
believes  this  change  in  the  kidney  to  be  induced  by  the  chemical 
poison,  and  he  explains  the  symptoms  of  the  disease  as  follows : 
The  cyanosis  is  a  consequence  of  the  arterial  contraction,  the  first 
effect  of  the  poison.  The  muscular  contractions  also  result  from  the 
action  of  the  toxin.  The  serous  exudate  of  the  intestines  follows 
upon  epithelial  necrosis.  Anuria  and  the  subsequent  symptoms 
appear  when  the  formation  and  absorption  of  the  poison  become 
greatest. 

Hueppe  states  that  the  severe  symptoms  of  cholera  can  be  ex- 
plained only  on  the  supposition  that  the  bacilli  produce  a  chemical 
poison,  which  resembles  muscarin  in  its  action. 

Villiers  isolated  by  the  Stas-Otto  method  from  two  bodies  dead 
from  cholera  a  poisonous  base  which  was  liquid,  pungent  to  the  taste, 
and  possessed  the  odor  of  hawthorn.  It  was  strongly  alkaline,  and 
gave  precipitates  with  the  general  alkaloidal  reagents.  From  one  to 
two  milligrammes  of  this  substance  injected  into  frogs  caused  de- 
creased activity  of  the  heart,  violent  trembling,  and  death.  The 
heart  was  found  in  diastole  and  full  of  blood,  and  the  brain  slightly 


56  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

congested.  However,  the  presence  of  this  substance  in  the  bodies  of 
persons  who  have  died  of  cholera  does  not  prove  that  its  production 
is  due  to  the  cholera  bacillus. 

Pouchet  extracted  from  cholera  stools,  with  chloroform,  an  oily 
base  belonging  to  the  pyridin  series.  It  reduced  ferric  as  well  as 
platinum  salts,  and  formed  a  readily  decomposable  hydrochlorid.  It 
was  a  violent  poison,  irritating  to  the  stomach,  and  retarding  the 
action  of  the  heart.  Subsequently,  he  obtained  a  similar  substance 
from  cultures  of  the  comma  bacillus. 

Brieger  used  pure  cultures  on  beef  broth,  which  was  rendered 
alkaline  by  the  addition  of  a  three  per  cent,  soda  solution.  These 
were  kept  at  from  37°  to  38°,  and  after  twenty-four  hours,  cadaverin 
was  found  to  be  present.  Older  cultures  yielded  very  small  quan- 
tities of  putrescin,  but  blood  serum  cultures  yielded  much  larger 
amounts  of  this  base.  While  cadaverin  and  putrescin  cannot  be  said 
to  be  poisonous,  they  cause  necrosis  of  tissue  into  which  they  are  in- 
jected, and  their  formation  by  the  cholera  germ  may  account  for  the 
necrotic  areas  observed  in  the  intestine  after  death  from  this  disease. 
The  lecithin  of  the  beef  broth  was  slowly  acted  upon  by  the  germ, 
and  with  age  the  amount  of  cholin  increased,  reaching  its  maximum 
during  the  fourth  week.  Creatin  proved  more  resistant  to  the  action 
of  the  bacillus ;  but,  after  six  weeks,  a  considerable  quantity  of 
creatinin  was  isolated,  and  a  similar  amount  of  methyl-guanidin. 
The  latter  is  highly  poisonous,  and  causes  muscle  tremors  and  dysp- 
noea. The  presence  of  methyl-guanidin  indicates  that  the  comma 
bacillus  acts  as  an  oxidizing  agent,  since  creatin  yields  this  substance 
only  by  oxidation.  In  addition  to  the  above-mentioned  ptomams, 
which  are  common  products  of  putrefaction,  Brieger  found  two 
poisons  which  he  at  that  time  considered  as  specific  toxins  of  the 
comma  bacillus.  One  of  these,  found  in  the  mercuric  chlorid  pre- 
cipitate, is  a  diamin,  resembling  trimethylendiamin.  It  caused  mus- 
cle tremor  and  cramps.  The  other  poison,  found  in  the  mercury 
filtrate,  produced  in  mice  a  lethargic  condition ;  the  respiration  and 
heart's  action  became  slow,  and  the  temperature  sank  so  that  the 
animals  felt  cold.  Sometimes  there  was  bloody  diarrhoea.  It  is 
quite  certain  that  the  above-mentioned  substances,  neither  singly  nor 
combined,  constitute  the  specific  toxin  of  the  cholera  bacillus. 

Brieger  and  Frankel  found  in  cultures  of  the  cholera  bacillus  an 
insoluble  proteid,  which,  when  suspended  in  water  and  injected  sub- 
cutaneously  in  guinea-pigs,  caused  death  after  from  two  to  three 
days.  Section  showed  inflammatory  swelling  and  redness  of  the 
subcutaneous  tissue,  extending  into  the  muscles,  for  some  distance 
about  the  point  of  injection,  but  no  necrosis.  There  was  no  change 
in  the  intestine  and  no  effusion  into  the  peritoneum.  In  some  in- 
stances there  was  beginning  fatty  degeneration  in  the  liver.  Upon 
rabbits  this  substance,  even  in  large  doses,  was  without  effect. 


ASIATIC  CHOLERA.  57 

Gamaleia  employed  cultures  which  had  been  sterilized  at  120°. 
Subcutaneous  injections  of  these  caused  transient  oedema  from  which 
the  animals  soon  recovered.  When  the  cultures  were  sterilized  at 
60°,  large  doses  (10  c.c.  per  kilogram  body-weight)  caused  death. 
In  this  connection  Bouchard  remarks  that  in  1884  he  obtained  by 
the  intravenous  injection  of  the  urine  of  a  cholera  patient  in  rabbits, 
muscular  tremor,  cyanosis,  albuminuria  and  diarrhoea,  but  that  he 
has  never  succeeded  in  inducing  these  symptoms  with  the  cholera 
vibrio. 

Petri  states  that  the  comma  bacillus  produces  in  pepton  cultures 
large  amounts  of  tyrosin  and  leucin,  a  small  quantity  of  indol,  fatty 
acids,  poisonous  bases,  and  a  poisonous  proteid.  The  proteid  re- 
sembles pepton  in  its  behavior  to  heat  and  chemical  reagents  and 
is  designated  by  Petri  as  "  toxopepton."  In  quantities  of  0.36 
gram  per  kilogram,  it  is  fatal  to  guinea-pigs  within  eighteen  hours, 
producing  muscle  tremor  and  paralysis.  Autopsy  shows  an  effu- 
sion into  the  peritoneal  cavity,  marked  injection  of  the  blood  vessels 
of  the  intestines,  and  isolated  hemorrhagic  spots.  It  is  possible  that 
the  substance  contains  the  cholera  toxin,  but  the  greater  part  of  it 
consists  of  harmless  proteid  bodies. 

Scholl  has  studied  the  chemical  products  of  the  cholera  bacillus 
when  grown  under  anaerobic  conditions.  For  this  purpose  he  em- 
ployed fresh  sterilized  eggs,  after  the  method  of  Hueppe.  The  inoc- 
ulated eggs,  after  being  kept  for  eighteen  days  at  36°,  were  opened. 
The  contents  smelled  intensely  of  hydrogen  sulphid,  but  not  of 
amines.  The  albumin  was  completely  fluid,  while  the  yolk  was  more 
solid  and  of  a  dark  color.  Five  c.c.  of  the  fluid  contents  injected  into 
the  abdomen  of  a  guinea-pig  caused  at  first  paralysis  of  the  posterior 
extremities,  then  general  paralysis,  and  death  within  forty  minutes. 
Section  showed  the  vessels  of  the  small  intestine  and  stomach  highly 
injected,  a  colorless  effusion  in  the  peritoneal  cavity,  and  the  heart 
in  diastole.  A  like  result  was  obtained  by  the  use  of  an  aqueous 
extract  of  a  precipitate  obtained  by  the  addition  of  the  albuminous 
content  of  the  egg  to  ten  times  its  volume  of  absolute  alcohol.  It  is 
more  than  probable  that  the  effect  obtained  in  these  experiments  was 
due  to  the  alcohol  or  hydrogen  sulphid  retained  in  the  albuminous 
substance. 

Hueppe  holds  that  the  cholera  poison  results  from  the  analytic  or 
ferment  action  of  the  germ  on  the  proteids  in  which  it  grows,  and 
that  the  proteids  of  the  bacterial  cells  are  not  poisonous.  Following 
the  classification  of  bacterial  proteids  which  we  have  made,  Hueppe 
would  place  the  cholera  toxin  among  our  bacterial  proteids  and  the 
immunizing  substance  among  the  cellular  proteids.  At  one  time  he 
claimed  that  these  substances  could  be  separated  in  the  following 
manner :  Rice-water  stools  from  cholera  patients  are  treated  with 
absolute  alcohol ;  both  the  toxin  and  the  immunizing  substance  are 


58  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

precipitated.  By  collecting  this  precipitate  and  extracting  it  with 
sterilized  water  or  physiological  salt  solution,  the  toxins  only  are  dis- 
solved. The  belief  that  a  separation  of  these  two  classes  of  proteids 
can  be  made  in  this  way  rests  upon  the  following  assumptions, 
neither  of  which  can  be  said  to  be  demonstrated  facts  :  (1)  The 
poison  and  the  immunizing  body  are  not  one  and  the  same  thing ; 
(2)  the  cellular  proteids  are  not  soluble  in  water  or  salt  solution. 

Hueppe  claims,  furthermore,  that  in  a  given  case  of  cholera  the 
toxin  may  be  formed  most  abundantly  and  the  immunizing  substance 
only  in  small  amount ;  in  such  a  case  the  symptoms  of  the  disease 
would  be  violent,  and  should  recovery  result,  immunity  to  subse- 
quent infection  would  be  slight.  With  the  conditions  reversed,  the 
disease  might  be  slight  and  the  immunity  established  great. 

Pfeiffer  and  his  students  have  held  that  the  cholera  toxin  is  an 
integral  part  of  the  bacterial  cell  and  that  it  is  not  set  free  or  capable 
of  manifesting  its  toxic  properties  until  the  germ  dies ;  on  the  other 
hand,  Metschnikoff,  Gruber,  Behring,  Ransom  and  others  contend 
that  the  living  cholera  germ  produces  both  in  vitro  and  in  vita  a 
soluble,  diffusible  poison.  It  is  not  necessary  to  go  into  detail  con- 
cerning this  discussion ;  but  we  will  give  a  brief  statement  of  some 
of  the  experiments  made  by  Metschnikoff  and  Roux.  A  collodion 
sac  of  about  4  c.c.  capacity,  filled  with  a  2  per  cent,  pepton  solution, 
and  inoculated  with  the  cholera  bacillus,  was  placed  in  the  abdominal 
cavity  of  a  guinea-pig.  A  second  animal  received  a  similar  sac  con- 
taining an  emulsion  of  cholera  bacilli  killed  by  chloroform  ;  while  a 
third  sac,  containing  pepton  solution  only,  was  placed  in  a  third  ani- 
mal. Guinea-pig  No.  3  remained  unaffected  ;  No.  2  showed  during 
the  first  few  days  some  slight  elevation  of  temperature  and  some 
emaciation ;  while  No.  1  died  after  from  three  to  five  days,  with 
symptoms  of  cholera  intoxication.  At  autopsy  the  typical  lesions  of 
cholera  infection  were  found  in  the  peritoneum,  intestines,  and  kid- 
neys ;  while  the  cholera  bacillus  could  not  be  found  in  the  peritoneal 
cavity,  in  the  blood,  nor  in  any  of  the  organs.  The  collodion  sac 
contained  a  large  culture  of  highly  motile  cholera  bacilli  but  no  leu- 
cocytes. In  order  to  obtain  the  toxin  in  artificial  cultures,  the  germ 
was  made  highly  virulent  by  passage  through  animals  and  by  sac 
cultures.  In  this  way  they  obtained  a  culture,  -z^^-^  c.c.  of  which  suf- 
ficed to  kill  guinea-pigs.  This  highly  virulent  germ  was  grown  in 
a  culture  medium  consisting  of  2  per  cent,  pepton,  1  per  cent,  com- 
mon salt,  and  2  per  cent,  gelatin  with  some  serum  added.  Cultures 
in  this  medium,  grown  at  37°  for  from  three  to  four  days  and  filtered 
through  porcelain,  killed  guinea-pigs  when  administered  subcuta- 
neously  in  the  proportion  of  one-third  c.c.  per  100-gram  body  weight. 
The  toxin  thus  obtained  was  not  materially  changed  on  being  boiled, 
but  did  lose  its  toxicity  on  contact  with  the  air,  especially  when  simul- 
taneously exposed  to  light.     Tubes  completely  filled  with  the  germ- 


ASIATIC  CHOLERA.  59 

free  filtrate  and  kept  in  the  dark  were  found  to  lose  only  one-third 
of  their  toxicity  after  six  months.  Guinea-pigs  were  found  to  be 
most  susceptible ;  rabbits  somewhat  more  resistant ;  mice  required 
relatively  twenty  times  the  dose  fatal  to  guinea-pigs ;  while  pigeons 
and  chickens  were  still  less  susceptible.  By  gradually  increased 
doses,  guinea-pigs,  rabbits,  goats,  and  horses  were  immunized  with 
this  toxin.  From  a  guinea-pig  thus  immunized  an  antitoxic  serum 
was  obtained,  and  it  was  found  that  one  c.c.  of  this  was  sufficient  to 
neutralize  four  c.c.  of  a  toxin,  two-thirds  c.c.  of  which  sufficed  to  kill 
a  guinea-pig  of  250  grams  in  14  hours.  By  treating  a  horse  for 
six  months  with  gradually  increased  doses  of  the  toxin  these  investi- 
gators secured  an  antitoxic  serum,  1  c.c.  of  which  was  sufficient  to 
neutralize  four  times  the  fatal  dose  of  the  toxin.  The  antitoxic 
serum  was  also  found  to  give  immunity  against  intra-peritoneal  in- 
fection with  living  cultures.  From  these  experiments  it  was  con- 
cluded that  in  the  treatment  of  cholera  an  antitoxic,  and  not  an 
anti-bacterial,  serum  is  needed.  It  should  be  stated  that  a  highly 
active  toxin  is  necessary  for  the  production  of  a  useful  serum. 

It  is  more  than  probable  that  the  cholera  toxin  is  formed  within 
the  cell  by  synthetical  processes,  and  that  it  readily  diffuses  through 
the  cell  wall.  On  this  assumption  both  Pfeiffer  and  Metschnikoff 
are  partially  right  in  their  contentions. 

Buj  wid  found  that  on  the  addition  of  from  5  to  10  per  cent,  hydro- 
chloric acid  to  bouillon  cultures  of  the  cholera  bacillus  there  is  de- 
veloped after  a  few  minutes  a  rose-violet  coloration,  which  increases 
during  the  next  half  hour  and  in  a  bright  light  shows  a  brownish 
shade.  The  coloration  is  more  marked  if  the  culture  be  kept  at  about 
37°.     In  a  pure  culture  this  reaction  does  not  occur.^ 

Brieger  found  that  this  color  is  due  to  an  indol  derivative.  In 
cholera  cultures  on  albumins  he  obtained  indol  by  distillation  with 
acetic  acid. 

Dunham  finds  the  best  medium  for  the  "  cholera  reaction  "  to  be 
a  one  per  cent,  alkaline  pepton  solution,  with  one-half  per  cent,  of 
common  salt.  Bujwid  prefers  a  two  per  cent.,  strongly  alkaline, 
pepton  solution  with  salt.  Jadassohn  finds  that  gelatin  cultures  give 
the  reaction  both  before  and  after  the  liquefaction  of  the  gelatin. 
The  undissolved  gelatin,  after  the  addition  of  hydrochloric  or  sul- 
phuric acid,  becomes  rose-violet.  Cohen  claims  that  cultures  of 
other  bacilli  give  a  similar  coloration,  but  Bujwid  explains  that  the 
results  obtained  by  Cohen  were  due  to  the  use  of  impure  acids, 
which  contained  nitrous  acid.  Salkowski  agrees  with  Bujwid,  and 
states  that  when  acids  wholly  free  from  nitrous  acid  are  used  the 
reaction  is  characteristic  of  the  comma  bacillus.     He  explains  the 

^Poehl  deserves  the  credit  of  being  the  first  to  call  attention  to  this  reaction, 
though  his  work  was  evidently  unknown  to  Bujwid  at  the  time  when  the  latter  pub- 
lished his  report. 


60  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

reaction  by  supposing  that  the  germ  produces  nitrous  acid,  which 
exists  in  the  culture  as  a  nitrite ;  on  the  addition  of  an  acid  the 
nitrous  acid  is  set  free  and  acting  upon  the  indol,  which  is  also  pres- 
ent, gives  the  coloration.  After  all,  wider  experience  has  shown 
that  Cohen  was  right  and  that  many  germs  respond  to  the  indol 
reaction. 

According  to  Hammurl,  cultures  of  the  germ  newly  taken  from 
stools  or  intestinal  content  do  not  give  the  indol  reaction  as  con- 
stantly as  those  germs  grown  on  artificial  media.  From  an  ex- 
haustive research  on  the  importance  of  this  test,  Petri  comes  to  the 
following  conclusions  :  (1)  Seven  pure  cultures  of  the  cholera  germ, 
from  as  many  sources,  gave  the  reaction  with  equal  distinctness. 
(2)  Of  one  hundred  other  bacteria  tested  in  the  same  way,  twenty 
gave  a  red  coloration.  In  nineteen  of  these  the  coloration  is  due  to 
the  nitroso-indol  reaction  of  Baeyer.  The  twentieth  (anthrax)  gave 
a  color  which  is  not  due  to  indol.  (3)  In  case  of  the  cholera  germ, 
and  the  others  as  well,  the  reaction  is  due  to  the  reducing  effect  of 
the  bacteria  on  nitrates.  It  is  most  marked  at  blood  temperature 
and  with  the  cholera  bacillus  ;  it  is  least  distinct  with  the  bacilli  of 
Finkler  and  Miller.  (4)  None  of  these  bacteria  convert  ammonia  into 
nitrite.  (5)  The  simple  addition  of  sulphuric  acid  is  sufficient  to  give 
the  test,  which,  however,  is  most  marked  when  the  nutrient  solution 
contains  0.01  per  cent,  nitrate.  (6)  The  reaction  is  most  marked  if 
the  sulphuric  acid  be  added,  after  the  addition  of  a  very  dilute 
nitrite  solution. 

Schuchardt  calls  attention  to  the  fact  that  Virchow  observed  a 
red  coloration  on  the  addition  of  nitric  acid  to  filtered  cholera  stools 
in  1846,  and  in  1885  Griesinger  made  mention  of  a  similar  obser- 
vation. 

A  "  cholera  blue  "  has  also  been  observed  by  Brieger  in  cultures 
in  meat  extract  containing  pepton  and  gelatin.  This  substance, 
which  is  yellow  by  refracted,  and  blue  by  transmitted,  light,  is  de- 
veloped by  the  addition  of  concentrated  sulphuric  acid  to  the  culture. 
It  may  be  separated  from  the  "  cholera  red  "  as  follows  :  Treat  the 
culture  with  sulphuric  acid,  then  render  alkaline  with  sodium  hy- 
drate and  extract  with  ether.  Evaporate  the  ether  and  remove  the 
"  cholera  red  "  with  benzol,  then  again  dissolve  the  "  cholera  blue  " 
in  ether.  The  characteristic  absorption  bands  for  this  coloring 
matter  begin  in  the  first  third  of  the  spectrum  between  E  and  F  and 
darken  all  of  the  zone  lying  beyond. 

Winter  and  Lesage  treat  a  bouillon  culture  of  the  cholera  germ 
with  sulphuric  acid,  dissolve  the  precipitate  in  an  alkaline  medium, 
reprecipitate  with  acid  and  redissolve  in  ether,  which  on  evaporation 
leaves  oily  drops,  and  these,  on  cooling,  form  a  yellow  mass  of  the 
appearance  of  a  fat.  This  substance  is  insoluble  in  water  and  acids, 
soluble  in  alkalis  and  ether.     It  melts  at  50°,  and  does  not  lose  its 


TETANUS.  61 

virulence  on  being  boiled  with  alcohol  rendered  feebly  alkaline. 
The  virulence  of  a  culture  and  the  amount  of  this  substance  con- 
tained therein  are  in  direct  proportion  to  each  other.  Small  doses 
(one  milligram  to  one  hundred  grams  of  body  weight)  in  feebly 
alkaline  solution,  introduced  into  the  stomachs  of  guinea-pigs  cause, 
as  a  rule,  within  from  four  to  six  hours,  a  chill  and  death  after 
twenty-four  hours.  With  larger  doses  the  temperature  falls  after 
from  one-half  to  one  hour  and  death  results  within  from  twelve  to 
twenty  hours.  Smaller  doses  cause  a  less  marked  reaction  and  the 
animal  recovers  within  twenty-four  hours.  Rabbits  succumb  only 
after  repeated  subcutaneous  injections.  The  substance  can  be  ex- 
tracted from  the  liver,  muscles,  kidneys,  and  urine  of  the  poisoned 
animals.  It  can  also  be  obtained  from  cultures  of  a  cholera  infantum 
germ.     It  is  quite  certain  that  this  substance  is  an  artificial  product. 

Tetanus. — Brieger  has  obtained  from  cultures  of  the  tetanus  germ 
four  poisonous  substances.  The  first,  tetanin,  which  rapidly  decom- 
poses in  acid,  but  is  stable  in  alkaline  solutions,  produces  tetanus  in 
mice  when  injected  in  only  a  few  milligrammes.  The  second,  teta- 
noxin,  produces  first  tremor,  then  paralysis,  followed  by  severe  con- 
vulsions. The  third,  to  which  no  name  has  been  given,  causes  teta- 
nus, accompanied  by  free  flow  of  the  saliva  and  tears.  The  fourth, 
spasmotoxin,  induces  clonic  and  tonic  convulsions.  The  same  in- 
vestigator isolated  tetanin  from  the  amputated  arm  of  a  man  with 
this  disease.  More  recent  researches  lead  us  to  attach  but  little 
importance  to  the  crystalline  bodies  discovered  by  Brieger,  and  it  is 
highly  probable  that  the  crystals  with  which  he  worked  were  not  of 
themselves  poisonous,  but  were  mixed  with  small  quantities  of  the 
toxin. 

In  their  researches  on  toxalbumins  Brieger  and  Frankel  obtained 
from  cultures  of  the  tetanus  germ  in  bouillon  containing  grape  sugar, 
a  substance  soluble  in  water,  which  when  injected  subcutaneously 
in  guinea-pigs  caused  tetanus  to  appear  after  about  four  days,  and 
led  to  a  fatal  termination. 

Later,  Brieger  and  Cohn  prepared  tetanus  poison  from  cultures  of 
the  bacillus  in  veal  broth  containing  one  per  cent,  of  pepton,  and 
one-half  per  cent,  of  common  salt.  These  cultures  were  rendered 
germ-free  by  filtration  through  porcelain,  and  treated  with  ammo- 
nium sulphate  to  supersaturation.  This  throws  the  poison  out  of 
solution  and  it  floats  on  the  surface,  from  which  it  is  removed  by  a 
platinum  spatula.  This  crude  poison,  when  dried  in  vacuo,  is  found 
to  contain  6.5  per  cent,  of  ammonium  sulphate.  Of  the  filtered  cul- 
ture 0.00005  c.c.  suffices  to  kill  mice.  From  one  liter  of  the  culture 
one  gram  of  the  dried  substance  was  obtained,  and  of  this  0.000,000,1 
gram  killed  a  mouse  with  the  typical  symptoms  of  tetanus.  This 
crude  product  contains,  besides  the  poison,  albumins,  pepton,  amido- 


62  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

acids,  volatile  substances,  and  ammonium  sulphate,  with  other  salts. 
The  albumin  was  removed  with  basic  lead  acetate.  The  pepton, 
amido-acids  and  salts  were  removed  by  dialysis,  and  finally,  evapo- 
ration in  vacuo  at  20-22°  removed  the  volatile  substances.  The 
toxin  thus  obtained  is  yellow,  flaky,  readily  soluble  in  water,  odor- 
less, and  similar  in  taste  to  gum  arable.  It  turns  polarized  light 
slightly  to  the  left.  It  fails  to  give  the  Millon  and  xanthoproteic 
reactions,  but  does  give  with  copper  sulphate  and  caustic  potash  a 
faint,  violet  coloration,  not  identical  with  the  rose  of  the  biuret  re- 
action. With  the  exception  of  ammonium  sulphate,  the  metallic 
salts,  as  sodium  chlorid  and  sulphate,  magnesium  sulphate,  potassium 
nitrate,  mercuric  chlorid  and  potassium  ferrocyanide  with  acetic  acid, 
fail  to  precipitate  the  purified  poison.  Moreover,  calcium  phosphate 
which  Roux  and  Yersin  used  for  carrying  down  the  diphtheria 
poison,  also  magnesium  carbonate  and  aluminum  hydrate,  do  not 
throw  the  tetanus  poison  out  of  solution.  The  toxin  thus  obtained 
contains  no  phosphorus  and  only  unweighable  traces  of  sulphur.  Of 
the  best  preparation  obtained  by  these  investigators,  0.000,000,05 
gram  killed  a  mouse  of  15  grams  weight.  The  authors  figure  from 
this  that  the  fatal  dose  for  a  man  of  70  kilograms  would  be  0.00023 
gram,  or  0.23  milligram,  and  0.04  milligram  would  induce  symptoms 
of  tetanus.  The  smallest  lethal  dose  of  atropin  for  an  adult  is  130 
milligrams,  and  of  strychnin  from  30  to  100  milligrams.  "  From 
this  one  can  judge  of  the  fearful  weapons  possessed  by  the  bacteria  in 
their  poisons." 

Fermi  and  Pernossi  draw  the  following  conclusions  from  their 
studies  of  the  tetanus  poison  :  (1)  Agar  cultures  are  the  most  poison- 
ous. Next  come  those  on  gelatin,  and  lastly  those  in  bouillon.  ^2) 
Chickens,  snakes,  turtles  and  tritons  are  immune  to  the  poison.  (3) 
In  the  above-mentioned  animals  this  toxin  may  remain  and  retain  its 
virulence  for  three  days,  and  even  longer.  (4)  Filtrates  from  agar 
and  gelatin  cultures  are  more  resistant  to  heat  than  those  from 
bouillon.  Like  the  enzymes,  the  purer  the  tetanus  poison  the  less 
stability  does  it  possess.  (5)  Dissolved  in  water,  the  tetanus  poison 
is  rendered  inert  by  a  temperature  of  55°,  but  in  the  dry  state  it  can 
be  heated  to  120°  without  loss  of  virulence.  (6)  When  the  dry 
poison  is  mixed  with  ether  or  chloroform  and  heated  to  80°,  it  is 
destroyed ;  but  with  amylic  alcohol  or  benzol,  a  temperature  of  1 00° 
is  required  to  accomplish  this  result.  (7)  Dissolved  in  water,  this 
poison  is  destroyed  by  direct  sunlight  after  an  exposure  of  eight  to 
ten  hours  (with  the  highest  temperature  on  a  blackened  thermometer 
at  56°)  and  after  fifteen  hours  when  the  temperature  does  not  exceed 
37°.  (8)  In  the  dry  state  the  tetanus  poison  can  be  exposed  to  the 
direct  sunlight  for  100  hours  without  loss  of  virulence.  (9)  Under 
the  action  of  an  electric  current  of  0.5  ampere,  continued  for  two 
hours,  the  substance  becomes  inert.     (10)  Gastric  juice  destroys  the 


TETANUS.  63 

poison  through  the  activity  of  the  hydrochloric  acid  and  not  by  vir- 
tue of  the  pepsin.  (11)  Ptyalin,  diastase,  and  emulsin  have  no  action. 
The  effect  of  trypsin  has  not  been  satisfactorily  determined.  (12) 
Putrefactive  germs  do  not  destroy  the  poison.  (13)  The  living,  but 
not  the  dead,  intestines  of  guinea-pigs  and  cats,  destroy  the  poison. 

(14)  The  chick  does  not  destroy  and  does   not  absorb  the  poison. 

(15)  The  poison  may  be  eliminated  by  the  kidneys  and  retain  its 
properties  in  the  urine.     (16)  The  poison  is  not  a  ferment. 

According  to  Ehrlich,^  the  tetanus  bacillus  produces  two  toxins, 
tetanospasmin  and  tetanolysin.  To  the  first  of  these  the  tetanic  con- 
vulsions are  due,  while  the  second  has  a  hemolytic  action.  Both  of 
these  toxins  are  present  in  the  precipitate  formed  by  the  addition  of 
ammonium  sulphate  to  bouillon  cultures  of  the  tetanus  bacillus,  but 
they  do  not  always  exist  in  the  same  proportion.  One  culture  may 
have  a  marked  tetanic  effect  and  manifest  but  little  hemolytic  action, 
while  a  second  culture  may  dissolve  red  blood  corpuscles  promptly 
but  show  only  slight  action  on  the  central  nervous  system.  These 
toxins  also  differ  in  their  stability,  the  hemolytic  poison  being  the 
less  stable  and  undergoing  complete  destruction  when  heated  to  50° 
for  twenty  minutes.  When  a  tetanus  culture  is  placed  in  a  menstruum 
containing  red  blood  corpuscles,  the  greater  part  of  the  tetanolysin 
combines  with  the  corpuscles  while  the  tetanospasmin  remains  in 
solution  ;  on  the  other  hand,  tetanospasmin  combines  with  nerve  cells, 
which  apparently  have  no  attraction  for  the  other  poison.  Each  ot 
these  substances  has  its  specific  antitoxin  and  in  the  blood  of  animals 
immunized  with  tetanus  cultures  one  or  the  other  antitoxin  may  pre- 
dominate. It  thus  happens  that  a  serum  may  be  strongly  antispastic, 
while  it  is  but  slightly  antilytic,  or  the  reverse  may  be  true. 

Madsen  ^  has  made  a  special  study  of  tetanolysin.  He  obtained  a 
mixed  toxin  by  precipitation  of  a  bouillon  culture  of  the  tetanus 
bacillus  with  ammonium  sulphate.  The  poison  thus  secured  was  of 
medium  strength,  the  fatal  dose  for  mice  of  15  grams  body  weight, 
being  0.000,001  gram.  This  poison  dissolves  the  red  blood  cor- 
puscles of  rabbits,  goats,  sheep,  horses  and  other  animals.  The 
blood  of  the  horse  and  that  of  the  rabbit  was  found  to  be  especially 
susceptible,  while  that  of  the  goat  was  less  so.  For  experimental 
purposes  the  defibrinated  blood  of  the  rabbit  diluted  with  0.85  per 
cent,  of  common  salt  solution  to  5  per  cent.,  was  used.  For  pur- 
poses of  demonstration  this  diluted  blood  was  placed  in  tall  test- 
tubes  of  Jena  glass.  This  gave  opportunity  for  the  observation  of 
the  effects  of  the  toxin  on  the  corpuscles  during  the  process  of  sub- 
sidence. To  the  tubes  thus  prepared  varying  quantities  of  the  poison 
were  added,  mixed,  allowed  to  stand  for  one  hour  at  37°,  and  then 
kept  over  night  at  a  low  temperature.     The  ultimate  result  was  ob- 

^  Berliner  klinische  Wochenschrift,  1898,  273. 
*  Zeitschrififur  Hygiene,  32,  214. 


64  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

served  twenty-four  hours  after  the  poison  was  added.  The  quantity 
of  the  poison  used  in  the  experiments  of  Madsen  varied  from  1.2 
c.c.  of  a  one  per  cent,  solution  to  0.2  c.c.  of  a  0.01  per  cent,  solution, 
and  a  control  tube  containing  no  toxin  served  for  contrast.  In  this 
way  the  effects  of  different  quantities  of  the  poison  were  made  plainly 
manifest  to  the  eye.  In  those  tubes  containing  the  larger  quantities 
of  the  poison  all  the  corpuscles  were  dissolved  and  there  remained 
a  blood-red  solution,  uniform  in  tint  from  top  to  bottom.  As  the 
amount  of  the  poison  was  diminished  the  number  of  undissolved 
corpuscles  collected  at  the  bottom  of  the  tube  was  increased  and  the 
color  of  the  supernatant  fluid  grew  less  intense  towards  the  top. 
With  the  larger  quantities  of  poison  the  destruction  of  corpuscles 
was  practically  instantaneous  and  occurred  before  there  was  time  for 
subsidence,  while  in  the  more  diluted  solutions  the  corpuscles  parti- 
ally subsided  before  they  were  acted  upon  by  the  toxin.  It  was  thus 
demonstrated  that  there  is  a  latent  period  in  the  action  of  the  poison 
and  that  this  period  increases  as  the  quantity  of  the  toxin  is  di- 
minished. It  was  also  found  that  lowering  the  temperature  increased 
for  a  given  amount  of  the  poison  the  latent  period.  It  was  also 
rendered  highly  probable  that  the  individual  corpuscles  vary  mark- 
edly in  their  resistance  to  the  hemolytic  action  of  the  poison. 
Furthermore,  it  was  shown  that  some  corpuscles  are  more  resistant 
or  less  resistant  to  this  toxin  than  to  other  hemolytic  poisons.  Thus, 
corpuscles  that  were  found  to  be  especially  susceptible  to  tetanolysin 
were  less  resistant  to  another  hemolytic  poison,  crotin. 

Madsen  continued  his  interesting  work  and  showed  that  the 
hemolytic  action  of  tetanolysin  can  be  neutralized  by  an  antitoxic 
serum.  In  these  experiments  he  used  a  tetanus  serum  each  gram  of 
which  contained  fifty  immunity  units  against  tetanospasmin.  This 
antitoxic  serum  was  diluted  to  one-half  per  cent,  with  a  mixture 
of  glycerin  and  common  salt  solution.  This  was  designated  as  his 
"  stock  solution  "  and  was  further  diluted  to  one-fortieth  of  one  per 
cent,  with  physiological  salt  solution  for  purposes  of  experimenta- 
tion. It  was  found  that  for  the  complete  neutralization  of  two  c.c. 
of  the  toxin  from  1.3  to  1.4  c.c.  of  the  -^-q-  per  cent,  antitoxin  solu- 
tion was  needed  ;  i.  e.,  when  a  mixture  containing  two  c.c.  of  toxin 
solution  and  1.3  c.c.  of  the  diluted  antitoxin  solution  was  added 
to  diluted  defibrinated  blood,  the  corpuscles  remained  unaffected. 
Following  the  method  pursued  by  Ehrlich  in  his  studies  of  the 
diphtheria  toxin,  Madsen  tried  the  effect  of  the  partial  neutralization 
of  the  tetanolysin  with  antitoxin.  As  a  result  of  these  experiments 
he  found  that  when  0.10  c.c.  of  the  antitoxin  solution  (which  is  one- 
thirteenth  of  the  amount  necessary  to  completely  neutralize  two  c.c. 
of  the  poison)  was  added  to  two  c.c.  of  the  poison  the  hemolytic 
action  of  the  toxin  was  decreased  to  one-half  its  value,  and  that 
when  0.25  c.c.  of  the  antitoxin  (one-fifth  of  the  amount  necessary 


TETANUS.  66 

for  complete  neutralization)  was  added  to  two  c.c.  of  the  toxin,  the 
latter  lost  nine-tenths  of  its  effect.  From  these  and  other  experi- 
ments Madsen  reaches  the  following  conclusions  : 

1.  In  cultures  of  the  tetanus  bacillus,  there  is  besides  tetanospasrain 
another  poison,  tetanolysin,  for  which  there  is  a  specific  antitoxin, 
which  may  be  designated  as  antilysin. 

2.  The  action  of  this  lysin  and  its  anti-body  can  be  measured  with 
great  exactness  by  methods  of  color  comparison. 

3.  Tetanolysin  combines  with  the  red  blood  corpuscles  and  the 
combination  is  preceded  by  a  latent  period  which  depends  upon  the 
amount  of  poison  and  the  temperature. 

4.  Investigation  shows  that  tetanolysin  presents  a  complicated 
neutralization  reaction  which  resembles  very  closely  that  of  the 
diphtheria  poison. 

5.  The  substance  consists  of  halves  which  differ  in  certain  funda- 
mental properties.  One  half  of  the  poison  consists  of  three  sub- 
stances, prototoxin,  deuterotoxin,  and  tritotoxin. 

6.  The  prototoxin  makes  up  only  one-third  of  the  quantity  of  the 
poison  but  possesses  one-half  of  the  hemolytic  action.  Like  the 
prototoxin  of  diphtheria  poison,  this  substance  is  readily  changed  to 
prototoxoid,  and  in  undergoing  this  transformation  it  loses  its  toxic- 
ity but  retains  its  combining  power. 

7.  The  deuterotoxin  makes  up  one-ninth  of  the  poison  but  pos- 
sesses two-fifths  of  the  total  hemolytic  action.  This  substance  re- 
sembles the  deuterotoxin  of  the  diphtheria  poison  and  is  relatively 
resistant  to  untoward  influences. 

8.  The  tritotoxin  makes  up  one-fourth  of  the  poison  but  possesses 
only  one-tenth  of  the  poisonous  action.  While  the  prototoxin  and 
deuterotoxin  act  both  under  high  and  low  temperatures,  the  trito- 
toxin has  no  action  at  a  temperature  below  10°.  Special  experi- 
ments show  that  the  tritotoxin  combines  with  the  red  blood  corpus- 
cles not  only  in  smaller  quantity  than  the  other  toxins,  but  that  its 
toxophorous  group  is  much  less  powerful  than  that  of  the  proto-  and 
deuterotoxins. 

9.  The  second  half  of  the  toxin  possesses  very  minute  toxicity 
and  is  known  as  toxon. 

10.  The  most  important  result  of  the  experiments  lies  in  the  de- 
termination that  in  tetanolysin,  as  in  diphtheria  toxin,  there  are  two 
groups,  a  haptophorous  group  by  which  the  substance  combines  with 
antitoxin,  and  a  toxophorous  group  by  which  the  combination  with 
the  red  blood  corpuscles  and  the  hemolytic  action  are  effected.  Of 
these  groups  the  first  one  is  relatively  stable  while  the  latter  is  easily 
modified  and  in  this  process  of  modification  the  toxin  becomes  a 
toxoid. 

11.  The  probabilities  are  that  tetanospasmin  has  a  neutralization 
structure  similar  to  that  possessed  by  tetanolysin. 

5 


66  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

By  separating  the  blood  corpuscles,  which  have  been  exposed  to 
the  action  of  varying  quantities  of  tetanolysin  for  different  periods 
of  time,  in  a  centrifuge,  and  subsequently  treating  them  with  tetanus 
antitoxin,  Madsen  ^  has  shown  that  the  poison  can  be  separated  from 
the  corpuscles  provided  a  sufficient  quantity  of  antitoxin  is  added 
before  hemolysis  occurs.  In  this  way  he  has  demonstrated  that  the 
antitoxin  may  act  as  a  curative  agent,  as  well  as  a  preventive. 

Ransom  ^  has  shown  that  both  the  toxin  and  antitoxin  of  tetanus 
pass  without  change  from  the  blood  into  the  lymph.  Shortly  after 
the  intravenous  injection  of  the  toxin  it  is  found  equally  distributed 
between  the  blood  and  the  lymph,  while  the  antitoxin  remains  in 
comparative  excess  in  the  blood.  From  these  observations  it  is  con- 
cluded that  the  toxin  behaves  like  inorganic  substances  in  passing 
from  the  blood  into  the  lymph,  while  the  antitoxin  resembles  more 
closely  the  proteids  in  this  respect. 

Bruschettini  has  studied  the  distribution  of  the  tetanus  poison  in 
the  body  and  its  elimination  ;  animals  poisoned  by  injections  of  the 
toxin  were  killed  just  before  death,  and  bits  of  various  organs  rubbed 
up  with  sterilized  water  were  injected  into  other  animals.  Emul- 
sions from  the  liver  and  suprarenal  capsule  were  invariably  without 
effect,  while  those  from  the  kidney  were  constantly  poisonous.  The 
blood  taken  from  the  vena  cava  was  found  to  be  poisonous  in  three 
out  of  four  experiments.  When  injections  were  made  under  the  skin 
the  lumbar  cord  was  active  in  four  out  of  eight  cases,  and  in  all 
when  the  injections  were  made  directly  into  the  sciatic  nerve.  When 
the  inoculations  were  made  under  the  dura  mater,  the  brain  was 
found  to  be  active,  while  the  lumbar  cord  remained  inactive ;  it  fol- 
lows from  this  that  the  toxin  not  only  circulates  in  the  blood,  but  is 
deposited  in  the  central  nervous  system.  This  author  has  also  shown 
that  tetanus  toxin  is  eliminated  by  the  kidneys.  Bruner  in  several 
cases  of  tetanus  in  man.  Stern  in  two,  and  Brieger  in  one  case,  were 
not  able  to  induce  tetanus  in  animals  by  injecting  even  large  amounts 
of  the  urine  of  their  patients.  This  does  not  cast  doubt  upon  the 
accuracy  of  the  report  of  Bruschettini  ;  it  only  shows  that  the  poison 
is  not  in  all  cases  eliminated  by  the  kidney  in  sufficient  quantity 
to  render  the  urine  highly  toxic.  In  a  fatal  case,  Vulpius  failed  to 
induce  tetanus  with  the  urine  voided  during  life,  but  succeeded  with 
that  found  in  the  bladder  after  death. 

Donitz^  has  shown  that  tetanus  toxin  combines  almost  instan- 
taneously with  the  cells  of  the  central  nervous  system.  In  his  ex- 
periments he  employed  twelve  times  the  minimum  lethal  dose  of  toxin, 
and  injected  this  directly  into  a  vein  of  the  ear.  He  first  determined 
the  amount  of  antitoxin  which,  mixed  with  this  quantity  of  toxin  in 

•  Zeitichrift  fiir  Hygiene,  32,  239. 

'^  Zeitschrlft  fur  physiol.  Chem.,  29,  1900. 

3  Deutsche  med.   Wochenschrift,  1897,  428. 


TETANUS.  67 

vitro,  was  necessary  for  complete  neutralization.  When  the  toxin  in 
this  amount  was  injected  intravenously  and  the  corresponding  amount 
of  antitoxin  injected  into  the  same  vein  of  the  opposite  ear  within 
two  minutes,  the  animals  invariably  died  of  tetanus.  In  order  to  save 
his  animals  treated  with  this  amount  of  toxin  it  was  found  necessary 
to  nearly  double  the  dose  of  antitoxin  when  injected  within  two 
minutes ;  while  if  four  minutes  elapsed  between  the  injection  of  the 
toxin  and  the  antitoxin,  six  times  the  quantity  necessary  for  neutrali- 
zation in  vitro  was  required,  and  when  this  time  was  extended  to 
one  hour,  twenty-four  times  this  amount  was  necessary.  Of  course, 
in  the  disease  the  toxin  is  generated  slowly  and  in  relatively  small 
amounts ;  therefore,  the  proportion  between  toxin  and  antitoxin 
necessary  to  neutralize  the  effects  of  the  former  is  not  found  to  be  so 
great.  The  same  experimenter  demonstrated  that  when  tetanus  is 
induced  by  inoculation  with  the  bacillus,  the  antitoxin  may,  experi- 
mentally at  least,  serve  as  a  curative  agent.  He  has  also  shown  that 
animals  treated  with  very  small  quantities  of  tetanus  toxin  may  die 
of  marasmus,  and  thus  there  may  be  such  a  thing  as  tetanus  siTie 
tetano.     This  has  been  observed  in  some  other  infectious  diseases. 

Wassermann  and  Takaki,  ^  have  shown  that  tetanotoxin  combines 
with  nervous  tissue,  forming  a  compound  which,  when  injected  into 
susceptible  animals,  is  found  to  be  inert.  They  prepared  a  solution 
of  tetanotoxin,  to  which  an  equal  part  of  glycerin  was  added  for  pres- 
ervation purposes.  This  solution  was  of  such  strength  that  one 
thousandth  of  a  cubic  centimeter  sufficed  to  kill  mice  in  the  course  of 
three  days.  Quantities  of  this  solution,  containing  from  one  to  ten 
fatal  doses  were  rubbed  up  into  emulsions  with  the  substance  of  the 
spinal  cord  or  brain,  and  this  mixture  was  injected  subcutaneously 
into  mice.  As  a  control,  equal  quantities  of  tissue  from  the  liver, 
kidney,  spleen,  and  bone  marrow  were  mixed  in  the  same  way  with 
the  tetanotoxin  and  likewise  injected  into  mice.  As  a  further  control, 
corresponding  amounts  of  tetanus  poison  without  admixture  with  any 
tissue  were  injected.  In  the  preparation  of  the  emulsions  the  organs 
were  taken  from  animals  immediately  after  death  and  rubbed  up  in 
a  mortar  with  physiological  salt  solution.  For  the  cord  from  a 
guinea-pig,  3  c.c.  of  salt  solution  were  used,  and  for  the  brain  from 
the  same  animal,  10  c.c.  were  employed.  By  these  experiments  it 
was  conclusively  demonstrated  that  the  normal  cord  and  brain  form 
an  inert  compound  with  the  toxin  of  tetanus,  while  none  of  the  other 
organs  has  any  such  effect.  It  was  found  from  about  two  hundred 
experiments  that  one  cubic  centimeter  of  the  brain  emulsion  neutral- 
izes ten  fatal  doses  of  the  tetanus  toxin,  and  has  a  marked  inhibitory 
action  on  several  times  this  amount.  The  antitoxic  action  of  the 
cord  was  found  to  be  less  marked  than  that  of  the  brain  substance. 
This  is  somewhat  unexpected,  inasmuch  as  the  symptoms  manifested 
1  Berliner  klinische  Wochenschriji,  35,  1898. 


68  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

in  tetanus  are  supposed  to  be  referable  to  the  action  of  the  toxin  on 
the  cord  rather  than  on  the  brain.  These  experiments  were  repeated 
with  brain  and  cord  substance  obtained  from  pigeons,  rabbits,  horses, 
and  men  with  practically  the  same  results  in  all  instances.  By  means 
of  the  centrifuge,  the  brain  emulsion  with  the  toxin  was  separated 
into  a  deposit  and  a  clear  supernatant  fluid,  and  it  was  demonstrated 
by  this  means  that  the  combined  toxin  and  brain  substance  was  to 
be  found  in  the  deposit.  In  other  words,  it  was  shown  that  the 
supernatant  fluid  had  no  antitoxic  action.  The  same  was  found  to 
be  true  of  fluids  obtained  from  the  ventricles  of  the  brain.  These 
experiments  seem  to  demonstrate  that  chemical  combination  takes 
place  between  the  toxins  introduced  into  the  body  and  the  formed 
cells,  and  that  solution  of  both  substances  entering  into  the  compound 
is  not  essential. 

Roux  and  Borrel,  ^  have  demonstrated  by  a  series  of  carefully  con- 
ducted experiments  that  small  doses  of  tetanus  toxin  when  injected 
directly  into  the  brain  substance  kill  animals,  while  much  larger 
quantities  of  the  same  poison  are  required  when  the  injection  is  made 
subcutaneously  or  intravenously.  This  undoubtedly  is  due  to  the 
fact  that  when  intracerebral  injection  is  made,  all  of  the  toxin  im- 
mediately combines  with  the  side  chains  of  the  brain  cells,  inflicting 
upon  this  tissue  an  injury  to  which  the  animal  speedily  succumbs. 

According  to  Ledantec,  the  poisonous  arrows  of  the  natives  of  the 
New  Hebrides  are  prepared  as  follows  :  The  points,  which  are  usu- 
ally made  from  human  bones,  are  first  covered  with  a  vegetable  resin, 
then  smeared  with  the  slime  of  swampy  places. 

Roncali  has  tested  forty  different  germs,  some  of  them  pathogenic 
and  others  non-pathogenic,  in  endeavoring  to  find  one  which  would 
neutralize,  either  by  its  growth  in  the  body  or  by  the  action  of  its 
products,  the  tetanus  poison.  His  results  were  wholly  negative. 
The  tetanus  poison  was  found  to  act  more  energetically  in  animals 
inoculated  with  other  bacteria  or  treated  with  their  products,  and  in 
no  case  was  there  any  evidence  of  antagonism  in  action. 

Diphtheria. — In  1887,  Loeffler  attempted  to  ascertain  the  nature 
of  the  diphtheria  toxin.  A  flask  of  bouillon  containing  pepton  and 
grape  sugar  was,  three  days  after  it  had  been  inoculated  with  the 
bacillus,  evaporated  to  10  c.c.  and  this  was  injected  into  an  animal, 
but  was  without  effect.  A  second  flask  of  the  same  material  was 
extracted  with  ether,  but  this  extract  was  also  found  to  be  inert. 
Next,  some  neutral  beef  broth  was  extracted  with  glycerin  four  or 
five  days  after  it  had  been  inoculated  with  the  bacillus.  The  glycerin 
extract,  when  treated  with  five  times  its  volume  of  absolute  alcohol, 
deposited  a  voluminous,  flocculent  precipitate,  which  was  collected, 
washed  with  alcohol,  dried  and  dissolved  in  water.  Further  precip- 
'  Annales  de  Ulnstitut  Pasteur,  12,  1898. 


DIPHTHERIA.  69 

itation  with  alcohol  and  a  current  of  carbonic  acid  gas,  secured  a 
white  substance,  and  the  injection  of  from  0.1  to  0.2  gram  of  this 
substance  subcutaneously  in  guinea-pigs,  caused  marked  pain,  fol- 
lowed by  a  fibrinous  swelling  with  hemorrhages  into  the  muscles  and 
oedema,  terminating  in  necrosis.  From  these  studies  Loeffler  con- 
cluded that  the  poison  belongs  to  the  enzymes. 

Roux  and  Yersin  found  that  bouillon  cultures  from  which  the 
bacillus  had  been  removed  by  filtration  through  a  Chamberland  filter 
are  poisonous,  especially  cultures  that  are  four  or  five  weeks  old. 
The  results  obtained  varied  with  the  amount  of  the  fluid,  the  species 
of  animal,  and  the  method  of  administration.  The  effects  observed 
were  a  serous  exudate  into  the  pleural  cavity,  marked  acute  inflam- 
mation of  the  kidney,  fatty  degeneration  of  the  liver,  especially 
after  injection  into  a  blood  vessel,  and  oedematous  swelling  in  the 
surrounding  tissue  after  subcutaneous  inoculation.  In  some  in- 
stances, paralysis,  generally  in  the  posterior  extremities,  followed. 
The  action  of  the  poison  was  slow,  and  death,  as  a  rule,  occurred 
days,  and  in  some  cases,  weeks  after  treatment,  and  was  preceded  by 
marked  emaciation.  The  cultures  first  employed  were  seven  days 
old ;  older  cultures  (six  weeks)  contain  more  of  the  toxin,  and  the 
symptoms  appear  within  a  few  hours.  In  cultures  especially  rich  in 
the  poison,  a  small  amount  (from  0.2  to  2  c.c.)  injected  under  the  skin 
in  guinea-pigs,  suffices  to  induce  the  symptoms.  Heating  to  100*^ 
for  twenty  minutes  renders  the  poison  inert,  and  a  temperature  of  58*^ 
maintained  for  two  hours  markedly  lessens  its  virulence.  The  toxin 
is  precipitated  by  absolute  alcohol,  and  is  carried  down  mechanically 
by  the  addition  of  calcium  chlorid  to  the  filtered  cultures.  The 
great  toxicity  of  this  substance  is  indicated  by  the  statement  of  Roux 
and  Yersin,  that  0.4  mg.  suffices  to  kill  eight  guinea-pigs  or  two 
rabbits  ;  and  that  0.02  g.  of  the  calcium  chlorid  precipitate,  containing 
about  0.2  mg.  of  the  pure  poison  will  kill  a  guinea-pig  within  four 
days. 

Brieger  and  Frankel  employed  cultures  of  bouillon  and  pepton 
containing  from  five  to  six  per  cent,  of  glycerin,  and  others  contain- 
ing two  per  cent,  of  blood  serum ;  the  latter  were  found  to  be  most 
suitable.  In  all  cases  they  confirmed  the  statement  of  Roux  and 
Yersin,  that  the  cultures,  at  first  alkaline,  become  strongly  acid,  and 
finally  again  alkaline,  with  the  exception  that  the  glycerin  cultures 
remain  acid.  For  the  removal  of  the  bacteria  two  methods  were 
employed.  First,  the  bacilli  were  destroyed  by  heat ;  when  a  tem- 
perature of  100°  was  employed  the  cultures  became  inert,  but  it  was 
found  that  exposure  for  from  three  to  four  hours  to  50°  was  sufficient 
to  destroy  the  germ,  while  the  virulence  of  the  toxin  was  not  affected. 
The  second  method  of  removing  the  bacteria  consisted  in  filtration 
through  a  Chamberland  filter.  The  germ-free  filtrate  could  be  heated 
to  50°  without  loss  of  toxicity,  while  a  temperature  of  60°  rendered 


70  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

it  inert.  In  the  majority  of  the  experiments  the  filtration  method 
was  used,  and  in  this  way  a  large  quantity  of  a  poisonous  fluid  of 
uniform  strength  was  obtained.  Varying  amounts  of  this  fluid  were 
used  upon  animals,  mostly  guinea-pigs  and  rabbits,  and  it  was  found 
that  the  effects  depended  upon  the  quantities  employed  and  the 
methods  of  administration.  In  all  cases  in  which  death  did  not  oc- 
cur too  early,  paralysis  appeared.  The  limbs  were  first  paralyzed, 
and  this  was  true  whether  the  fluid  was  administered  intravenously 
or  subcutaneously.  The  post-mortem  appearances  were  identical 
with  those  observed  after  inoculation  with  the  bacillus,  with  the  ex- 
ception of  the  absence  of  the  pseudo-membrane.  After  subcutaneous 
injection  there  was  a  gelatinous,  grayish-white,  sometimes  reddish, 
oedematous  fluid,  formed  at  the  point  of  injection,  and,  after  large 
doses,  necrosis.  In  cases  in  which  death  was  delayed,  there  were 
effusions  in  the  pleura,  fatty  degeneration  of  the  liver,  and  inflam- 
mation of  the  kidneys. 

Brieger  and  Frankel  conclude  this  part  of  their  report  with  the 
following  statement :  "  We  have  shown  that  the  Loeffler  diphtheria 
bacillus  produces  in  its  cultures  a  poisonous,  soluble  substance,  sep- 
arable from  the  bacteria,  which  causes  in  susceptible  animals  the 
same  phenomena  induced  by  inoculation  with  the  living  micro-organ- 
ism. We  have  further  shown  that  this  substance  is  destroyed  by  a 
temperature  over  GO*',  but  that  it  can  be  heated  to  50°,  even  in  the 
presence  of  an  excess  of  hydrochloric  acid,  without  being  destroyed. 
This  last  fact  is  contrary  to  the  assumption  that  the  chemical  poison 
of  the  diphtheria  bacillus  is  a  ferment  or  an  enzyme." 

The  filtered  cultures  were  tested  for  basic  products,  but  with  neg- 
ative results,  excepting  that  small  amounts  of  creatinin  and  cholin 
were  found.  They  were  also  distilled  at  from  20°  to  35°  in  a  vacuum, 
but  the  distillate  was  found  to  be  inert.  The  toxin  is  soluble  in 
water,  insoluble  in  alcohol,  and  non-dialyzable.  It  is  precipitated 
by  saturation  with  ammonium  sulphate.  As  a  result  of  their  first 
work  on  this  subject,  Brieger  and  Frankel  concluded  that  the  diph- 
theria toxin  is  a  toxalbumin ;  however,  they,  as  well  as  others,  have 
since  learned  that  while  the  body  obtained  by  them  contained  the 
toxin,  the  bulk  of  it  was  made  up  of  other  substances. 

Taugl  has  shown  experimentally  that  the  toxin  is  formed  in  the 
body  as  well  as  in  culture  flasks.  A  large  piece  of  pseudo-membrane 
was  macerated  in  an  ice-chest  in  water  for  twenty-four  hours,  and 
then  filtered  through  porcelain.  The  filtrate,  injected  into  animals, 
produced  all  the  symptoms  that  had  been  obtained  by  similar  em- 
ployment of  artificial  cultures.  The  same  observer  found  that  in 
some  cases  in  which  the  animals  were  inoculated  with  the  sterilized 
culture  through  the  mucous  membrane  a  pseudomembrane  formed  at 
the  point  of  injection.  Diphtheria  toxin  has  also  been  found  in  the 
tissues,  blood,  and  urine. 


DIPHTHERIA.  71 

While  crude  diphtheria  toxin,  in  the  form  of  sterilized  cultures  of 
the  bacillus,  has  been  used  in  securing  immunity  and  in  the  produc- 
tion of  antitoxin,  which  has  proved  to  be  of  such  priceless  value  in 
the  treatment  of  the  disease,  no  one  has  up  to  the  present  time  been 
able  to  isolate  the  poison,  and  we  remain  ignorant  of  its  chemical 
constitution. 

From  a  large  number  of  most  carefully  conducted  experiments 
with  the  toxin  and  antitoxin  of  diphtheria,  Ehrlich  ^  has  formulated 
a  theory  concerning  the  constitution  of  the  former.  This  theory  has 
undergone  several  modifications  since  it  was  first  proposed,  and  it  is 
difficult  to  give  an  exact  statement  of  it  as  it  now  stands.  However, 
we  will  attempt  to  state  in  condensed  form  its  essential  points  as 
follows  :  ^ 

1.  Toxins  and  antitoxins  neutralize  one  another  after  the  manner 
of  chemical  reagents.  The  chief  reasons  for  this  belief  lie  in  the  ob- 
served facts  (a)  that  neutralization  takes  place  more  rapidly  in  con- 
centrated than  in  dilute  solutions,  and  (6)  that  warmth  hastens  and 
cold  retards  neutralization.  From  these  observations  Ehrlich  con- 
cludes that  toxins  and  antitoxins  act  as  chemical  reagents  do  in  the 
formation  of  double  salts.  A  molecule  of  the  poison  requires  an 
exact  and  constant  quantity  of  the  antitoxin  in  order  to  produce  a 
neutral  or  harmless  substance.  This  implies  that  a  specific  atomic 
group  in  the  toxin  molecule  combines  Avith  a  certain  atomic  group  in 
the  antitoxin  molecule. 

2.  The  antitoxin  is  a  reaction  product  of  the  living  organism  and 
not  a  transformation  product  of  the  toxin  introduced  in  securing  im- 
munity. It  is  thought  that  when  the  toxin  is  introduced  into  the 
animal  body  in  small  quantities  it  combines  with  certain  side-chains 
in  the  molecules  of  the  living  cells.  These  side-chains  are  supposed 
to  be  necessary  for  the  proper  functioning  of  the  cells,  which,  finding 
themselves  deprived  in  part  of  their  function  on  account  of  combi- 
nation with  the  toxin,  produce  other  and  similar  atomic  groups  or 
side-chains,  and  these  being  formed  more  rapidly  than  they  are 
taken  up  by  the  toxin,  are  cast  off  into  the  blood  and  constitute  the 
antitoxin.  For  instance,  when  a  small  quantity  of  tetanus  toxin  is 
introduced  into  the  animal  body  it  combines  with  certain  side-chains 
in  the  molecules  of  the  cells  of  the  central  nervous  system  and 
renders  these  atomic  groups  useless  so  far  as  the  function  of  the  cell 
is  concerned.  The  cell,  in  order  to  compensate  for  its  loss,  produces 
another  side-chain  similar  to  the  one  of  which  it  has  been  deprived. 
Being  called  upon  repeatedly  to  exercise  this  activity,  there  is  not 
only  compensation,  but  over-compensation,  and  the  result  is  that  more 
side-chains  are  formed  than  the  cell  can  use,  and  these  break  off  and 

^  Die  Wertbemessung  des  Diphtheriaheilserums  und  deren  theoretische  Gnind- 
lagen. 

''For  a  more  detailed  statement  of  Ehrlich' s  theory  see  Chapter  VII. 


72  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

float  away  in  the  blood,  constituting  the  antitoxin.  Moreover,  the 
atomic  group  or  side-chain,  after  being  liberated  from  the  cell,  may 
possibly  acquire  greater  avidity  for  combination  with  the  toxin ;  or, 
in  other  words,  the  toxin  will  combine  more  readily  with  these  side- 
chains  when  free  and  floating  in  the  blood  than  when  they  constitute 
parts  of  the  molecules  of  the  cells. 

3.  The  diphtheria  toxin,  as  it  exists  in  sterilized  cultures,  is  com- 
posed of  equal  parts  of  toxin  and  toxon.  The  toxon,  which  accord- 
ing to  this  theory  is  supposed  to  exist  in  the  diphtheria  culture,  is 
believed  to  be  without  any  serious  eifect  upon  animals.  It  may 
cause  local  oedema,  but  never  kills.  Ehrlich's  theory  supposes  that 
the  toxon,  which  we  may  regard  as  inert,  has  quantitatively  the  same 
power  of  combination  with  antitoxin  as  is  possessed  by  the  toxin, 
but  combines  with  the  antitoxin  with  less  avidity.  In  order  to 
understand  why  the  theory  provides  for  the  existence  of  toxin  and 
toxon  in  equal  quantities  in  the  diphtheria  culture,  we  will  use  cer- 
tain formulae  proposed  by  Ehrlich  : 

T=  the  minimum  fatal  dose  of  the  toxin  (a  quantity  sure  to  kill 
a  guinea-pig  of  250  grams  weight  within  five  or  six  days). 

1=  one  immunity  unit  which  is  capable  of  neutralizing  the  efi*ects 
of  100  T. 

Now,  if  I  be  mixed  with  100  T  and  injected  into  an  animal,  no 
ill  efi'ect  results.  If  the  toxin  were  a  simple  body,  I  plus  100  T 
plus  T  should  kill  one  animal.  But  experimentally  we  find  that 
this  amount  has  no  effect,  and  must  be  greatly  increased  before  there 
is  enough  free  toxin  in  the  mixture  to  kill.  Ehrlich  represents  the 
quantity  of  poison  necessary  to  neutralize  /  by  X^.  Then  I  plus  L^ 
is  a  mixture  which  contains  neither  free  toxin  nor  free  antitoxin  and 
is  wholly  without  effect  upon  animals.  By  L_^_  he  indicates  the  quan- 
tity of  poison  which  must  be  added  to  I  in  order  to  kill  a  guinea-pig 
of  250  grams  weight  within  five  or  six  days.  Let  us  suppose  that 
in  a  given  culture  T  =  0.01  c.c,  then  J  =  1.00  c.c.  =  100  T.  L^  = 
1.00  c.c.  =  100  T.  Now,  if  this  were  a  poison  like  strychnia  we 
would  expect  that  i^  =  1.01  c.c.  =  101  T,  would  kill,  but  in  reality, 
as  has  been  stated,  we  find  that  we  have  to  add  more  of  the  toxin  to 
L^  in  order  to  produce  fatal  results  with  the  mixture,  and  we  have 
theoretically  the  following : 

T=  0.01  c.c. 
L^  =  2.01  c.c.  =  201  T. 
L^=  1.00  c.c.  =  100  T. 
I)=  1.01  c.c.  =  101  T. 

As  here  figured  out,  Ehrlich's  theory  which  provides  for  the  ex- 
istence in  the  crude  toxin  of  equal  parts  of  toxin  and  toxon,  is  the 
only  possible   explanation.     In  L^  both  toxin  and  toxon  are  fully 


DIPHTHERIA.  73 

combined  with  antitoxin.  When  more  toxin  is  added  the  antitoxin 
is  dissociated  from  the  toxon  and  combines  with  the  toxin  because 
it  has  greater  affinity  for  the  latter  substance.  This  continues  until 
all  the  antitoxin  is  separated  from  the  toxon  and  combined  with  the 
toxin,  and  there  can  be  no  free  toxin  until  this  point  is  reached. 
Practically,  however,  while  the  difference  between  i^  and  L^  is  always 
more  than  1,  it  is  never  as  great  as  101.  Therefore,  we  must  con- 
clude that  while  there  is  reason  for  believing  that  there  exists  in 
diphtheria  cultures  substances  corresponding  to  Ehrlich's  toxin  and 
toxon,  the  facts  do  not  justify  the  statement  that  these  bodies  exist 
in  equal  quantities.  Moreover,  as  has  been  pointed  out  by  Park  and 
Atkinson,^  the  standard  for  T  is  arbitrary  and  it  might  have  been 
placed  at  the  amount  necessary  to  kill  a  guinea-pig  of  250  grams 
within  ten  days,  or  to  cause  death  from  paralysis  within  two  or  three 
weeks.  The  same  critics  state  :  "  Even  if  the  toxin  molecule  be 
divided  as  he  (Ehrlich)  believes,  still  in  any  given  bouillon  there  is 
actual  destruction,  as  well  as  production,  going  on  all  the  time  of  all 
substances,  not  only  those  toxic  to  guinea-pigs,  but  also  of  those 
which,  though  non-toxic,  still  neutralize  antitoxin." 

4.  It  is  observed  frequently  that  while  the  toxicity  of  a  given 
sterilized  culture  decreases  on  standing,  the  quantity  of  antitoxin 
which  it  will  neutralize  remains  constant.  This  observation  has 
caused  Ehrlich  to  provide  in  his  theory  for  the  toxoids  which  have 
already  been  mentioned  (p.  35). 

Flexner  ^  has  studied  quite  minutely  and  exhaustively  the  patho- 
logical lesions  induced  by  certain  bacterial  toxins.  Concerning  the 
gross  appearances  after  death  from  subcutaneous  or  intraperitoneal 
injections  of  sterilized  cultures  of  the  diphtheria  bacillus,  he  makes 
the  following  statement :  "  Much  oedema,  sometimes  accompanied 
with  small  extravasations  of  blood  and  often  of  an  exquisite  gelati- 
nous type,  was  noted.  The  axillary  and  inguinal  glands  were 
always,  often  greatly,  enlarged  and  were  congested  or  thickly  dotted 
with  hemorrhages  within  the  substance  of  the  capsule  and  the  glandu- 
lar tissues.  The  corresponding  lymph  glands  on  the  opposite  side 
were  also  enlarged,  but  usually  less  so  than  those  on  the  side  of  the 
inoculation.  This  enlargement  involved  the  superficial  and  deep 
sets.  The  cervical  glands  along  the  carotids  and  trachea  to  the 
maxillae  also  showed  an  increase  in  size.  The  thyroids  were  without 
exception  deep  brownish-red  in  color.  The  thymus  gland  presented 
a  rosy,  and  sometimes,  owing  to  hemorrhages,  a  speckled  appearance  ; 
the  bronchial  and  pericardial  glands,  often  very  difficult  to  find  in 
the  healthy  animal,  were  quite  prominent ;  the  mediastinal  glands 
were  swollen  and  congested  or  ecchymotic.  The  mesenteric  and 
retroperitoneal  glands  were  also  enlarged,  and  sometimes  considerably 

^  Expenmental  Medicine,  3,  513. 

^  Johns  Hopkins  Hospital  Reports,  6,  259. 


74  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

softened.  The  patches  of  Peyer  in  the  intestine  at  times  showed  up 
with  a  prominence  sufficient  to  enable  them  to  be  detected  through 
the  serosa  of  the  intestine.  Tlie  peritoneal  cavity  often  contained  an 
excess  of  clear  fluid  ;  hydrothorax  also  occurred,  and  in  rare  instances 
reached  such  a  high  degree  as  to  completely  compress  both  lungs,  the 
apices  alone  being  spared  and  insufflated.  The  pericardial  sac  was 
also  unduly  distended  with  a  clear  serous  fluid.  The  adrenal  glands 
presented  a  reddish  color  externally,  and  on  section  the  medulla  ap- 
peared deeply  congested  or  even  hemorrhagic.  The  kidneys  were 
often  pale.  The  liver  was,  as  a  rule,  congested  and  exhibited  upon 
its  surface  and  within  its  substance  a  variable  number  of  yellowish 
or  yellowish-white  areas,  some  not  larger  than  a  pin-point,  others  2 
mm.  in  extent,  which  were  usually  surrounded  by  a  hyperemic  zone. 
In  some  cases  a  few  of  such  foci  could  be  discovered  with  the  naked 
eye  ;  in  others  they  were  almost  innumerable.  The  spleen,  as  a  rule, 
was  not  greatly  enlarged,  it  was  more  often  pale  than  congested,  and 
the  Malpighian  bodies  showed  distinctly.  The  lungs  varied  in  ap- 
pearance, the  difference  depending  apparently  not  so  much  upon  any 
pathological  alterations  of  which  they  were  the  seat,  but  rather  upon 
the  quantity  of  fluid  present  in  the  pleural  cavities.  The  heart  was 
pale,  the  chambers,  especially  of  the  right  side,  were  distended  with 
dark,  clotted  blood.  Subserous  hemorrhages,  especially  in  the  peri- 
toneal cavity,  were  an  interesting  but  variable  feature  of  the  disease." 
The  same  observer  made  a  close  microscopical  study  of  the  various 
tissues  of  animals  treated  with  crude  diphtheria  toxin.  In  the 
lymph  glands,  the  earliest  changes  were  found  to  consist  of  swelling 
of  the  nucleus  and  perhaps  also  of  the  protoplasm.  Under  larger 
doses  of  the  poison  the  nuclei  were  often  observed  to  be  homogeneous 
and  seemed  converted  into  granular  balls  of  variable  size,  staining 
evenly  and  intensely  but  exhibiting  a  metachromatic  reaction. 
Under  still  larger  doses,  the  nuclei  were  observed  to  undergo  disin- 
tegration. The  fragmentation  was  irregular,  indicating  the  powerful 
effect  of  the  toxin.  In  some  cases  the  detritus  was  seen  within  the 
protoplasm  of  the  original  cell,  while  in  others  the  fragments  had 
become  free,  owing  to  the  destruction  of  the  cell  body.  Necrobiotic 
foci,  such  as  have  been  described  by  Oertel  after  death  from  diph- 
theria, were  observed  in  the  lymph  glands  in  certain  cases.  The 
spleen  was  generally  hyperemic,  the  excess  of  blood  being  contained 
within  the  sinuses  of  the  pulp.  The  earliest  changes  were  found  to 
cxjnsist  of  swelling  of  the  cells  of  the  Malpighian  bodies.  Mitotic 
division  was  observed  in  the  cells  but  did  not  seem  to  progress 
actively.  Pathological  alterations  were  found  in  the  pulp  affecting 
the  framework  and  the  vascular  contents.  In  the  intestines,  the 
most  pronounced  changes  were  encountered  in  the  epithelium.  These 
alterations  consisted  of  proliferation  by  mitosis  and  of  degeneration. 
There  was  extensive  fragmentation   of  cells  within   the  crypts  of 


DIPHTHERIA.  76 

Lieberkiilm.  In  Peyer's  patches  the  changes  were  similar  to  those 
observed  in  other  lymphatic  structures.  There  was  no  evidence  of 
denudation  of  the  surface  in  any  part  of  the  alimentary  tract.  The 
mucosa  of  the  stomach  showed  only  insignificant  lesions.  The  sur- 
face of  the  liver  often  exhibited  small  yellow,  opaque  points  and 
lines  which  upon  section  were  found  to  extend  down  into  the  organ. 
These  points  and  lines  indicate  focal  degeneration  and  death  of  liver 
cells.  In  the  majority  of  instances  the  liver  was  congested  and 
showed  more  or  less  evidence  of  fatty  metamorphosis.  "  The  kid- 
neys were  not  invariably  the  seat  of  fatty  changes,  but  it  was  the 
rule  to  find  more  or  less  fat  (1)  in  the  convoluted  tubules  of  the 
labyrinth,  (2)  in  the  straight  tubules,  and  (3)  in  the  glomeruli.  In 
some  instances  the  fatty  metamorphosis  of  the  epithelial  cells  was 
extreme.  In  all  cases,  practically,  the  cells  were  much  swollen  and 
coarsely  granular,  the  proteid  granules  being  combined  with  fat.  In 
rare  instances  a  deposition  of  lime  salts  was  noted  in  the  straight 
tubules.  These  deposits  yielded  carbonic  acid  gas  on  the  addition 
of  strong  acids.  Among  the  most  interesting  changes  observed  was 
a  hyaline  transformation  of  the  glomerular  capsules  and  smaller 
arteries."  The  lesions  observed  in  the  lungs  were  confined  almost 
exclusively  to  the  vessels,  the  endothelial  cells  of  the  pulmonary 
branches  and  the  vesicular  capillaries  showed  fragmentary  degener- 
ation. The  adrenals  were  markedly  congested  with  occasional 
hemorrhages  into  the  tissues,  and  the  same  condition  was  observed 
in  the  thyroid  gland.  Fatty  metamorphosis  was  the  most  common 
pathological  condition  found  in  the  myocardium.  The  alterations 
observed  in  the  muscle  substance  of  the  heart  affected  both  the 
nuclei  and  the  protoplasm,  being  most  marked  in  the  former.  "  The 
earliest  and  most  common  appearance  consists  of  swelling  and  elon- 
gation of  the  nuclei  and  alteration  in  their  shape,  the  fibers  in  the 
meantime  showing  little  change.  Certain  nuclei  later  assume  a 
deeper  color,  owing  chiefly  to  an  intense  staining  of  three  or  four  or 
more  globular  bodies  in  their  interior ;  but  soon  afterwards  the 
nuclear  membrane  either  degenerates  or  becomes  invisible,  and  the 
bodies  become  free  and  appear  as  fragments.  The  substance  of  the 
fibers  belonging  to  the  affected  nuclei  has  in  the  meantime  quite  dis- 
appeared or  takes  on  a  swollen  and  an  attenuated  aspect." 

Councilman,  Mallory,  and  Pearce^  have  made  a  most  exhaustive 
study  of  the  gross  and  microscopical  lesions  found  in  man  after  death 
from  diphtheria.  Many  of  these  pathological  changes  are  evidently 
due  to  the  toxin  because  they  are  found  in  parts  of  the  body  not 
reached  by  the  bacillus.  In  the  lymph  nodes  the  most  characteristic 
lesion  was  found  to  consist  in  the  formation  of  discrete  foci  due  to 
cell  proliferation  combined  with  necrosis.     In  all  cases  they  observed 

'  Diphtheria,  A  Study  of  the  Bacteriology  and  Pathology  of  Two  Hundred  and 
Twenty  Fatal  Cases,  1901. 


76  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

lesions  in  the  kidney  varying  from  simple  degeneration  to  the  more 
serious  condition  of  acute  nephritis.  The  more  severe  forms  of  de- 
generation were  observed  in  cases  which,  on  account  of  the  intensity 
of  the  poison,  succumbed  shortly  after  the  onset  of  the  disease.  The 
lesions  in  the  human  liver  diflfer  from  those  produced  experimentally 
in  animals  chiefly  in  the  greater  frequency  of  the  central  situation  of 
the  necrosis.  The  changes  in  the  intestines  consist  principally  of 
hyperplasia  of  the  lymphoid  structures.  "  The  slight  extent  of  the 
lesions  does  not  indicate  the  action  of  toxins  absorbed  from  the  ali- 
mentary canal ;  they  are  probably  due  to  the  action  of  toxins  from 
the  blood  principally.  There  is  nothing  in  the  character  of  the  le- 
sions to  indicate  the  elimination  of  the  toxin  by  the  alimentary  canal." 
In  the  heart  fatty  degeneration  was  observed  in  severe  cases  of  short 
duration  and  more  extensive  degenerations  in  more  prolonged  cases. 
The  pathological  changes  found  in  the  heart  are  often  sufiicient  to 
account  for  the  impairment  of  this  organ  so  greatly  feared  in  this 
disease.  Thrombosis  occurs  frequently  and  is  due  to  primary  necro- 
sis of  the  endocardium.  The  changes  observed  in  the  lungs  in  man 
after  death  from  diphtheria  are  largely  due  to  other  microorganisms, 
among  which  the  pneumococcus  is  probably  the  most  important. 

Tuberculosis. — In  1865,  Yillemin  demonstrated  the  infectious 
character  of  this  disease  by  inducing  it  in  animals  by  feeding  them 
upon  tuberculous  sputum  and  tissues.  In  1868,  Chauveau,  and,  a 
few  years  later,  Cohnheim  experimentally  confirmed  the  discovery  of 
Villemin,  which  must  be  regarded  as  one  of  the  most  important  con- 
tributions to  medical  knowledge  made  during  the  nineteenth  century. 
In  1878,  Tappeiner  showed  that  tuberculosis  might  be  transmitted 
by  the  inhalation  of  infected  dust.  However,  the  nature  of  the  in- 
fecting agent  in  tuberculous  tissue  remained  unknown  until  1882, 
when  Koch,  after  a  most  exhaustive  research  covering  different  mani- 
festations of  tuberculosis  in  man  and  some  of  the  lower  animals, 
announced  the  discovery  of  the  specific  bacterium  of  this  disease. 
This  work  was  so  thoroughly  done  that  practically  every  statement 
made  by  Koch  in  his  first  report  stands  to-day  unchallenged.  The 
twenty  years  that  have  elapsed  since  that  time  have  each  brought  its 
confirmations  of  the  facts  then  recorded.  It  is  not  within  the  prov- 
ince of  this  book  to  discuss  in  any  detail  the  bacterium  of  tubercu- 
losis, and  it  must  suffice  to  say  that  the  causal  relation  of  the  bacillus 
tuberculosis  to  the  disease  is  not  now  questioned  by  any  competent 
authority.  Every  case  of  tuberculosis  is  due  to  infection  from  a  pre- 
existing case  in  man  or  beast.  We  will  concern  ourselves  wholly 
with  the  chemical  poisons  produced  by  this  bacterium  and  by  virtue 
of  which  the  symptoms  of  the  disease  and  death  are  induced.  We 
may  be  permitted,  however,  to  call  attention  to  the  fact  that  in  its 
later  stages  tuberculosis  becomes  a  mixed  infection  and  the  chemical 


TUBERCULOSIS.  77 

products  of  more  than  one  bacterium  constitute  the  causal  factors  in 
this  form  of  poisoning. 

Koch's  tuberculin,  with  which  he  hoped  to  cure  the  disease,  is  the 
crude  poison  formed  by  his  bacillus  and  is  known  as  tuberculin. 
The  methods  of  preparing  this  substance  have  varied  somewhat  and 
we  will  mention  some  of  them.  Koch  prepared  tuberculin  in  the 
following  manner  :  Meat  infusion  containing  one  per  cent,  of  pepton 
and  from  four  to  six  per  cent,  of  glycerin  is  placed  in  sterilized 
flasks  with  broad  bottoms.  The  flasks  are  only  partially  filled  in 
order  that  the  surface  of  the  fluid  should  be  as  great  as  possible. 
A  small  mass  of  a  growth  of  tubercle  bacilli  is  taken  from  a  culture 
on  glycerin  agar  or  blood  serum,  and  floated  on  the  surface  of  the 
meat  infusion  in  the  flask,  which  is  then  placed  in  an  incubator  at 
37°.  The  bacilli  grow  abundantly  on  the  surface  of  the  meat  infu- 
sion, forming  a  thick,  yellowish-white  layer.  After  about  six  weeks, 
growth  stops,  the  bacterial  layer  begins  to  break  into  pieces  and  these 
fall  to  the  bottom  of  the  flask.  The  culture  is  now  evaporated  to 
one-tenth  its  volume  on  the  water- bath.  The  concentration  increases 
the  per  cent,  of  glycerin  to  from  forty  to  fifty,  and  this  ingredient 
prevents  the  growth  of  extraneous  bacteria  and  renders  the  fluid  per- 
manent for  an  indefinite  time.  After  filtration  through  porcelain, 
this  fluid  constitutes  the  crude  tuberculin  of  Koch.  It  will  be  seen 
that  it  must  contain,  in  addition  to  the  water  and  glycerin,  any  other 
soluble,  unchanged  constituent  of  the  original  meat  infusion,  any 
split  products,  if  there  be  such,  arising  from  the  cleavage  action  of 
the  bacilli  on  the  components  of  the  culture  medium,  and  all  soluble 
constituents  of  the  bacterial  cells.  The  toxin  in  this  impure  form 
is  not  destroyed  by  the  temperature  of  the  water-bath.  Ultimate 
analyses  of  the  crude  tuberculin  have  been  made,  but  it  must  be 
evident  from  what  has  just  been  said  concerning  the  complexity  of 
its  composition  that  such  determinations  are  without  value.  It  does 
not  contain  any  ptomain  or  other  basic  body. 

Bujwid  obtained  tuberculin  by  extracting  the  growths  of  the 
bacillus  on  glycerin  agar  tubes,  heating  to  100°  for  ten  minutes, 
filtering  through  porcelain  and  concentrating  at  a  low  temperature. 
As  thus  prepared,  the  fluid  resembles  very  much  the  preparation 
already  described. 

Tuberculin  may  also  be  obtained  from  bacilli  grown  on  potatoes. 
The  freshly  cut  surfaces  of  the  sterilized  potatoes  are  washed  with  a 
1  per  cent,  sterilized  solution  of  sodium  bicarbonate,  then  mois- 
tened with  sterilized  water  containing  five  or  six  per  cent,  of  glyce- 
rin. On  potatoes  thus  prepared  the  bacillus  grows  abundantly  at 
a  temperature  of  37°.  After  further  development  has  ceased,  the 
growths  are  extracted  with  water  or  water  and  glycerin. 

In  1897  Koch^  reported  his  attempts  to  improve  the  preparation 
^  Deutsche  med.  Wochemchrift,  1897,  209. 


78  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES, 

of  tuberculin.  At  first  he  extracted  the  bacilli  with  a  decinormal 
solution  of  caustic  soda.  This  extract,  when  filtered  through  paper 
and  injected  into  animals,  induced  a  reaction  similar  to  that  which 
followed  the  use  of  the  original  preparation ;  but  it  was  found  that 
abscesses  were  likely  to  follow  the  employment  of  this  material.  It 
was  also  observed  that  it  contained  tubercle  bacilli  and  for  this 
reason  it  was  filtered  through  porcelain.  The  filtrate  was  germ-free, 
but  was  found  not  to  possess  the  reaction  of  tuberculin.  It  was  thus 
evident  that  filtration  through  porcelain  had  removed  not  only  the 
bacilli,  but  the  active  constituent  of  the  preparation.  In  his  further 
researches  with  the  bacillus,  Koch  was  able  to  extract  two  character- 
istic chemical  substances,  both,  of  which  belong  to  the  unsaturated 
fatty  acids.  One  of  these  is  freely  soluble  in  dilute  alcohol  and  is 
readily  saponified  with  alkalis,  while  the  other  is  soluble  only  in 
boiling  absolute  alcohol  and  saponifies  with  difficulty.  Both  of  these 
substances  take  the  so-called  tubercle  stain,  i.  e.,  they  are  stained  in- 
tensely red  with  carbolic  fuchsin  and  retain  this  color  on  treatment 
with  dilute  nitric  acid  and  with  alcohol.  Bacilli  from  which  these 
fatty  bodies  have  been  extracted  retain  their  form,  but  no  longer 
take  their  characteristic  stain. 

Finally,  Koch  prepared  his  improved  tuberculin  in  the  following 
manner  :  The  bacilli  of  fresh,  highly  virulent  cultures  are  dried  and 
rubbed  into  a  very  fine  powder,  which  is  then  added  to  distilled 
water  with  which  it  forms  a  colloidal  mixture.  This  aqueous  prep- 
aration is  placed  in  a  centrifuge  which  makes  four  thousand  revolu- 
tions per  minute,  and  after  half  an  hour  it  separates  into  an  upper, 
opalescent  but  transparent  layer,  which  contains  no  bacilli,  and  into 
a  deposit.  The  deposit  is  dried,  again  rubbed  up  in  a  mortar,  mixed 
with  water  and  centrifuged,  and  this  is  repeated  until  there  is 
obtained  a  perfectly  clear  fluid.  The  upper  layer  obtained  the  first 
time  in  the  centrifuge  Koch  designated  as  TO,  while  the  upper  layers 
obtained  by  subsequent  treatment  in  the  centrifuge  he  distinguished 
as  TR.  The  addition  of  50  per  cent,  glycerin  to  TO  causes  no 
change,  while  in  TR  it  produces  a  flocculent  white  precipitate.  This 
indicates  that  TR  contains  those  substances  which  are  insoluble  in 
glycerin,  while  TO  is  made  up  of  the  constituents  of  the  bacterial 
cells  soluble  in  this  reagent.  In  its  action  upon  man  and  other 
animals,  TO  resembles  the  original  tuberculin  while  TR  is  supposed 
to  have  the  curative  action  first  attributed  to  tuberculin  without  any 
of  its  ill-effects.  Koch  stated  that  with  TR  men  and  animals  could 
be  so  immunized  that  they  did  not  react  to  TO  or  crude  tuberculin. 
TR  is  preserved  with  the  addition  of  20  per  cent,  of  glycerin. 

De  Schweinitz  ^  isolated  from  liquid  cultures  of  the  bacillus  tuber- 
culosis a  crystalline  substance  having  a  melting  point  of  161°  to 
164°,  readily  soluble  in  ether,  alcohol  and  water.  Analysis  showed 
'  TransactioTis  of  the  Association  of  American  Physicians,  1897. 


TUBERCULOSIS.  79 

that  this  body  corresponds  closely  to  teraconic  acid  and  its  discoverer 
believes  that  the  necrotic  effects  observed  in  tuberculosis  are  due  to 
this  agent.  It  is  also  a  fever-reducing  substance,  while  the  albu- 
minoid obtained  from  cultures  of  the  tubercle  bacillus  causes  an  ele- 
vation of  temperature. 

Klebs '  states,  as  a  result  of  his  investigations,  that  the  tubercle 
bacillus  contains  two  fatty  bodies.  One  of  these  may  be  extracted 
with  ether ;  it  has  a  reddish  color,  melts  at  42°,  and  constitutes  20.5 
per  cent,  of  the  weight  of  the  bacillus.  The  other  fat  is  insoluble  in 
ether,  but  may  be  extracted  with  benzol ;  its  melting  point  has  not 
been  accurately  determined,  but  is  something  over  50°,  and  it  con- 
stitutes 1.14  per  cent,  of  the  substance  of  the  germ.  The  specific 
coloration  of  tubercle  bacillus  with  carbolic  fuchsin  is  due  to  the 
presence  of  these  fatty  bodies,  and  after  their  removal  the  bacilli, 
although  retaining  their  form,  fail  to  stain.  The  greater  part  of  the 
tubercle  bacillus,  after  the  removal  of  the  above-mentioned  fats,  con- 
sists of  a  nuclein,  which  may  be  purified  after  digestion  with  pepsin 
and  hydrochloric  acid,  by  solution  in  dilute  alkalis  and  precipitation 
with  alcohol.  In  this  way  Klebs  obtained  a  nuclein  which  yielded 
between  8  and  9  per  cent,  of  phosphorus.  The  third  important  con- 
stituent of  the  tubercle  bacillus  is  the  glycerin-water  extract,  which 
probably  consists  of  a  mixture  of  substances,  some  of  which  are  pro- 
teid  in  character. 

Euppel  *  thinks  that  there  are  three  kinds  of  fatty  substances  in 
the  tubercle  bacillus.  The  first  constitutes  about  8  per  cent,  of  the 
total  weight  of  the  organism,  from  which  it  can  be  removed  with  cold 
alcohol.  During  the  process  of  extraction  the  alcohol  becomes  in- 
tensely red,  and  this  is  supposed  to  be  due  to  a  chromogen 
contained  in  the  bacillus,  which  develops  into  the  coloring  matter  on 
exposure  to  air.  On  evaporation  of  this  alcoholic  extract  there  re- 
mains a  smeary  mass,  consisting  largely  of  free  fatty  acid.  When 
the  free  fatty  acids  are  removed  by  the  ordinary  method  of  treatment 
with  soda  solution  and  ether,  the  mass  obtained  on  the  evaporation 
of  the  ether  melts  between  55  and  60°.  This  substance  is  easily 
saponified,  and  contains  along  with  the  free  fatty  acids  another  sub- 
stance soluble  in  ether,  which  is  believed  to  be  one  of  the  higher 
alcohols.  The  second  form  of  fat  contained  in  the  bacillus  may  be 
extracted  from  the  residue  left  after  extraction  with  cold  alcohol,  by 
means  of  hot  alcohol.  This  substance  begins  to  liquefy  at  65°,  but 
does  not  become  altogether  clear  until  a  temperature  of  200°  is 
reached.  The  hot  alcoholic  extract  saponifies  with  difficulty,  and 
appears  to  consist  of  fatty  esters  of  the  higher  alcohols.  The  third 
fatty  substance  may  be  removed  by  means  of  ether.  It  melts  at  from 
65  to  70°,  and  on  being  heated  gives  off  an  odor  similar  to  that  of 

^  Centralblatt  f.  Bakteriologie,  20/488. 
'  Zeitschrift  f.  physiol.  Chemie,  26,  1899. 


80  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

beeswax.  The  total  amount  of  fatty  substances  contained  in  the 
tubercle  bacillus  varies  from  8  to  26  per  cent. 

According  to  Ruppel,  the  soluble  albuminous  substances  contained 
in  the  tubercle  bacillus  are  best  obtained  by  the  action  of  superheated 
steam  on  bacilli  previously  deprived  of  their  fatty  content.  The 
procedure  recommended  is  as  follows  :  The  bacilli  are  first  extracted 
with  a  1  per  cent,  soda  solution  or  heated  with  a  dilute  solution  of 
glycerin,  thoroughly  extracted  with  alcohol  and  ether,  dried  and 
rubbed  into  a  fine  powder,  which  is  then  treated  with  ten  times  its 
volume  of  5  per  cent,  glycerin  solution  and  kept  in  the  autoclave  at 
150°  for  about  two  hours.  The  extract  thus  obtained  is  filtered 
while  still  hot,  and  it  is  found  that  the  filtrate,  at  first  clear,  forms  a 
deposit  on  standing,  and  complete  separation  may  be  obtained  in  the 
centrifuge.  The  soluble  proteids  obtained  in  this  way  constituted 
from  18  to  20  per  cent,  of  the  total  weight  of  the  bacilli,  and  con- 
sisted exclusively  of  albumoses  analogous  to  the  atmidalbumose  of 
Neumeister.  The  solution  of  mixed  atmidalbumoses  is  dark  in 
color  and  may  be  precipitated  by  ammonium  sulphate,  sodium 
chlorid,  or,  best  of  all,  with  absolute  alcohol  after  the  addition  of  a 
small  amount  of  sodium  chlorid  and  hydrochloric  acid.  On  drying, 
the  alcoholic  precipitate  is  found  to  consist  of  a  light  powder,  which, 
as  has  been  stated,  constitutes  about  20  per  cent,  of  the  fat-free 
bacilli. 

Ruppel  finds  that  when  tubercle  bacilli  have  been  finely  broken 
up,  they  give  up  about  half  their  substance  to  aqueous  extracts. 
When  finely  divided  tubercle  bacilli  are  shaken  in  water  there  is 
formed  a  milky  emulsion,  and  this,  when  placed  in  the  centrifuge, 
separates  into  a  transparent,  feebly  opalescent,  yellowish  fluid,  and  a 
deposit.  When  this  deposit  is  dried  and  weighed  it  is  found  to  con- 
stitute about  half  the  weight  of  the  powdered  bacilli  originally  taken. 
The  reaction  of  the  fluid  portion  is  generally  feebly  alkaline,  but 
may  be  neutral.  It  contains  no  coagulable  proteid,  and  of  the 
general  color  reactions,  it  responds  only  to  the  biuret  test.  Acetic 
acid  produces  a  considerable  precipitate,  which  is  not  soluble  in 
excess,  but  which  dissolves  in  dilute  alkalis,  from  which  it  may  be 
precipitated  anew  on  the  addition  of  acids.  The  acetic  acid  precipi- 
tate contains  a  little  more  than  4  per  cent,  of  phosphorus.  It 
responds  to  neither  the  Millon  nor  the  xanthoproteic  test,  but  the 
biuret  reaction  is  positive.  When  dissolved  in  glacial  acetic  acid 
and  warmed  with  sulphuric  acid,  it  does  not  give  a  red  coloration. 
About  25  grams  of  this  substance  was  shaken  with  1  per  cent,  sul- 
phuric acid,  and  the  acid  extract  thus  obtained  was  precipitated  by 
the  addition  of  absolute  alcohol.  The  precipitate  was  found  to  con- 
sist of  a  snow-white,  flocculent  powder,  and  the  amount  obtained 
was  0.7  of  a  gram.  It  is  soluble  in  warm  water,  from  which  it 
partially  deposits  on  cooling.     When  dissolved  in  warm  water,  the 


TUBERCULOSIS.  81 

addition  of  barium  hydrate  precipitates  barium  sulphate,  which 
may  be  removed  by  filtration  and  the  filtrate  precipitated  with 
alcohol.  The  substance  thus  obtained  does  not  respond  to  any  of 
the  color  reactions  for  proteids  with  the  exception  of  the  biuret  test. 
Ruppel  believes  it  to  be  a  protamin,  and  proposes  for  it  the  name, 
tuberculosamin.  He  believes  that  in  the  tubercle  bacillus  this 
protamin  is  combined  with  nucleinic  acid,  and,  indeed,  from  the 
part  insoluble  in  1  per  cent,  sulphuric  acid  he  obtained  a  nucleinic 
acid  which  contains  9.42  per  cent,  of  phosphorus.  He  proposes 
that  the  nucleinic  acid  in  the  tubercle  bacillus  be  known  as  tubercu- 
linic  acid. 

According  to  Behring,^  tuberculinic  acid,  as  prepared  by  Ruppel, 
contains  a  histon-like  body,  on  the  removal  of  which  the  tubercu- 
linic acid  is  obtained  chemically  pure.  Behring  states  that  the 
nucleinic  acid  obtained  from  tubercle  bacilli,  or  tuberculinic  acid, 
is  quite  different  from  all  other  nucleinic  acids.  To  this  substance 
he  attributes  the  toxic  action  of  tubercle  bacilli,  and  he  states  that 
one  gram  of  tuberculinic  acid  is  capable  of  destroying  the  life  of 
600  grams  of  normal  guinea-pig  when  injected  subcutaneously,  and 
90,000  grams  when  introduced  intra-cerebrally,  and  that  the 
same  amount  of  tuberculinic  acid  is  capable  of  destroying  the  life  of 
60,000  grams  of  guinea-pig,  already  infected  with  tuberculosis,  when 
administered  subcutaneously ;  and  40,000,000  grams  of  the  same 
tissue  when  injected  intra-cerebrally.  In  distinguishing  physiolog- 
ically between  tuberculinic  and  other  nucleinic  acids,  tuberculous 
animals  should  be  selected  on  account  of  their  greater  susceptibility 
to  the  tuberculinic  acid. 

Levene,^  agrees  with  Ruppel  that  the  tubercle  bacillus  contains 
both  free  and  combined  nucleinic  acid,  and  the  following  is  his 
statement  concerning  the  method  of  obtaining  both  of  these  :  "  With 
the  view  of  obtaining  the  free  nucleic  acid,  neutralized,  dried  and 
pulverized  bacilli  were  repeatedly  extracted  with  a  5  per  cent, 
sodium  chlorid  solution,  and  an  8  per  cent,  ammonium  chlorid  solu- 
tion. The  extracts  obtained  were  then  treated  with  picric  acid  and 
acidulated  with  acetic  acid.  To  the  filtrate  from  this  precipitation 
alcohol  was  added,  and  the  precipitate  thus  formed  redissolved  in 
water  and  reprecipitated  with  alcohol.  The  perfectly  white  pre- 
cipitate was  redissolved  in  water,  slightly  acidulated  with  acetic 
acid,  and  treated  with  a  solution  of  cupric  chlorid.  The  precipi- 
tate thus  formed  was  washed  with  water  until  copper-free ;  then 
with  alcohol  until  chlorin-free ;  finally  with  ether  and  then  dried  in 
vacuo  over  sulphuric  acid,  and  in  air-bath  at  105°  to  constant 
weight.  .  .  .  The  residue  after  the  sodium  chlorid  treatment  was 
treated  for  two  hours  with  a  4  per  cent,  solution  of  sodium  hydrate 

^  Berliner  Minische  Wochenschrift,  36. 
^  Journal  of  Medical  Research,  1,  1901. 


82  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

and  some  sodium  acetate,  then  neutralized  with  acetic  acid.  An  ex- 
cess of  picric  acid  was  then  added  and  then  rendered  acid  with 
acetic  acid.  To  the  filtrate  alcohol  was  added  and  the  precipitate 
thus  fijrmed  was  redissolved  and  reprecipitated.  The  precipitate  was 
biuret-free  and  possessed  all  the  properties  of  nucleic  acids.  On 
heating  with  mineral  acids,  it  did  not  reduce  Fehling  solution.  It 
was  again  redissolved  in  water  by  the  aid  of  some  alcohol,  the  solution 
was  then  rendered  acid  with  acetic  acid,  and  the  copper  salt  of  the 
nucleic  acid  obtained  as  above." 

By  this  method  Levene  obtained  from  samples  of  tubercle  bacilli 
grown  on  different  culture  media,  nucleinic  acids  which  varied  in 
phosphorus  content  from  6.58  to  13.9  per  cent.  The  same  investi- 
gator obtained  a  glycogen-like  substance  from  the  tubercle  bacillus. 
This  gives  an  opalescent  solution,  which  on  the  addition  of  iodin 
colors  similarly  to  glycogen.  On  being  heated  with  mineral  acids 
it  reduces  Fehling  solution.  The  method  of  obtaining  this  carbohy- 
drate is  given  as  follows  :  "  The  bacilli  were  treated  as  described 
above  for  the  separation  of  nucleic  acid.  The  picric  acid  filtrate 
was  then  treated  with  alcohol.  The  precipitate  thus  formed  con- 
sisted of  nucleic  acid  and  glycogen.  The  acid  can  be  removed 
on  addition  of  a  solution  of  cupric  chlorid.  The  copper  compound 
of  the  glycogen  remains  in  solution  and  from  the  latter  it  can  be 
precipitated  by  the  addition  of  alcohol.  The  copper  compound 
thus  obtained  is  dissolved  in  water,  the  solution  is  acidulated 
with  hydrochloric  acid  until  the  solution  reacts  acid  to  congo,  and 
the  glycogen  is  then  precipitated  with  alcohol.  Should  the  precipi- 
tate still  contain  some  copper,  the  latter  can  be  removed  by  repeat- 
ing the  last  operations." 

Koch's  first  announcement  concerning  tuberculin  awakened  great 
activity  in  the  study  of  the  chemistry  of  the  bacillus  of  this  disease. 
Maffucci  found  that  cultures,  when  grown  upon  glycerin  or  blood 
serum  for  from  one  to  six  months,  and  then  sterilized  by  being  re- 
peatedly heated  to  from  65°  to  70°,  produced  in  guinea-pigs,  when 
employed  subcutaneously,  a  progressive  marasmus,  which  terminated 
fatally  within  from  fourteen  days  to  six  months.  He  also  found  that 
eggs  inoculated  with  sterilized  cultures  of  the  chicken  tuberculosis 
bacillus  produced  young  which  were  feeble  and  soon  died  of  emacia- 
tion. In  neither  the  guinea-pigs  nor  chickens  could  he  find  any 
tubercles.  Crookshank  and  Herroun  reported  the  isolation  of  a 
ptomain  and  an  albumose  not  only  from  artificial  cultures  of  the 
bacillus,  but  also  from  bovine  tuberculous  tissue.  Both  of  these 
bodies  were  said  to  cause  an  elevation  of  temperature  in  tuberculous, 
and  a  depression  in  healthy  animals.  Zuelzer  reported  the  isolation 
of  a  poisonous  ptomain  from  agar  cultures  of  the  bacillus  tuberculo- 
sis. The  injection  of  one  eg.  or  less  of  this  substance  subcutaneously 
in  rabbits  or  guinea-pigs  caused,  after  from  three  to  five  minutes. 


TUBERCULOSIS.  83 

increased  frequency  of  respiration  (to  1 80  per  minute  ?)  and  an  ele- 
vation of  temperature  of  from  0.5°  to  1°.  Marked  proti^usio  hulbi 
was  a  constant  symptom  and  the  pupils  were  dilated.  From  two  to 
three  eg.  sufficed  to  kill  rabbits,  death  occurring  in  from  two  to  four 
days.  The  place  of  injection  was  found  to  be  reddened  and  hemor- 
rhagic spots  were  observed  in  the  mucous  membrane  of  the  stomach 
and  small  intestines.  Occasionally  considerable  amounts  of  clear 
fluid  were  found  in  the  peritoneal  cavity.  As  early  as  1888,  Ham- 
merschlag  had  ascertained  that  cultures  of  the  tubercle  bacillus  are 
toxic  to  certain  animals.  Subsequently  he  reported  that  almost  27 
per  cent,  of  the  cellular  substance  of  the  bacillus  tuberculosis  is  solu- 
ble in  alcohol  and  ether.  In  this  extract,  he  found,  in  addition  to 
fat  and  lecithin,  a  poison  which  induced  in  rabbits  and  guinea-pigs 
convulsions  followed  by  death.  The  part  of  the  germ  insoluble  in 
alcohol  and  ether  was  found  to  consist  of  cellulose  and  proteids. 
Weyl  obtained  by  macerating  the  bacillus  in  dilute  soda  solution  an 
extract  which,  when  injected  into  animals,  caused  local  necrosis. 
Prudden  and  Hodenpyl  summarized  the  results  which  they  obtained 
by  the  inoculation  of  animals  with  dead  tubercle  bacilli,  as  follows  s 
"  These  dead  tubercle  bacilli  are  markedly  chemotactic.  When  in- 
troduced in  considerable  amount  into  the  subcutaneous  tissue  or  into 
the  pleural  or  abdominal  cavities,  they  are  distinctly  pyogenetic, 
causing  aseptic,  localized  suppuration.  Under  these  conditions  they 
are  capable,  moreover,  of  stimulating  the  tissues  about  the  suppurative 
foci  to  the  development  of  a  new  tissue,  closely  resembling  the  diffijse 
tubercle  tissue  induced  by  the  living  germ.  We  have  found  that 
dead  tubercle  bacilli  introduced  in  small  numbers  into  the  blood 
vessels  of  the  rabbit  largely  disappear  within  a  few  hours  or  days, 
but  that  scattering  individuals  and  clusters  may  remain  here  and 
there  in  the  lungs  and  liver,  clinging  to  the  vessel  walls  for  many 
days  without  inducing  any  marked  changes  in  the  latter.  After  a 
time,  however — earliest  in  the  lungs,  later,  as  a  rule,  in  the  liver — 
a  cell  proliferation  occurs,  in  the  vicinity  of  these  dead  germs,  which 
leads  to  the  formation  of  new,  multiple,  nodular  structures,  bearing 
a  striking  morphological  resemblance  to  miliary  tubercles.  There  is 
in  them,  however,  no  tendency  to  cheesy  degeneration  and  no  evi- 
dence of  proliferation  of  the  bacilli,  but  rather  a  steady  diminution 
in  their  number.  It  seems  to  us  that  the  new  structures  regenerate 
in  a  proliferation  of  the  vascular  endothelium  under  the  stimulus  of 
the  dead  and  disintegrating  germs."  Babes  and  Broca  showed  that 
the  introduction  of  dead  tubercle  bacilli  into  the  bodies  of  animals 
rendered  them  susceptible  to  the  action  of  tuberculin,  and  the  re- 
action was  most  marked  when  the  tuberculin  had  not  been  extracted 
from  the  bacilli.  They  also  claimed  that  the  local  changes  induced 
by  the  injection  of  dead  bacilli  were  improved  and  could  be  healed 
by  injections  of  tuberculin  ;  but  that  under  the  influence  of  the  tuber- 


84  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

culin  the  dead  bacilli  were  often  carried  from  the  place  of  deposit  and 
distributed  by  means  of  the  circulation  to  different  parts  of  the  body 
where  subsequently  tubercular  nodules  formed. 

The  physiological  action  of  tuberculin  is  so  pronounced  and  at  first 
was  believed  to  be  so  markedly  sui  generis,  that  its  discoverer,  and 
through  him  the  greater  part  of  the  medical  world,  was  for  a  short 
time  led  into  the  belief  that  a  specific  and  sure  cure  for  the  greatest 
plague  of  man  had  been  found.  The  grounds  for  this  belief  were 
founded  principally  upon  the  following  effects  observed  in  the  action 
of  tuberculin  :  (1)  Small  doses,  one  mg.  or  even  less,  injected  subcu- 
taneously  into  individuals  suffering  from  tuberculosis  caused  marked 
elevation  of  temperature  ;  while  similar  doses  injected  in  the  same  way 
into  non-tubercular  persons  produced  no  appreciable  effect.  More- 
over, persons  sick  with  other  diseases  than  tuberculosis  were  not 
found  to  be  especially  susceptible  to  the  action  of  tuberculin.  Here, 
then,  is  a  substance  that  has  a  specific  action,  a  chemical  body  by  the 
effects  of  which  one  can  distinguish  a  tubercular  from  a  non-tuber- 
cular individual.  If  all  the  cows  of  a  large  herd  be  treated  with 
tuberculin  in  proper  amount,  and  a  record  of  the  temperature  be 
made  for  twenty-four  hours  before  and  for  the  same  length  of  time 
after  the  treatment,  it  will  be  found  that  in  some  a  febrile  reaction — 
an  elevation  of  one  degree  or  more  in  temperature — occurs,  while  the 
temperature  of  others  remains  unaffected,  or  is  but  slightly  changed. 
Now,  if  all  these  animals  be  killed  and  examined,  it  will  be  found 
that  those  that  have  manifested  the  febrile  reaction  are  tuberculous ; 
while  those  that  failed  to  react  to  tuberculin  are  not  tuberculous. 
A  similar  test  to  this  was  made  by  Koch  on  tuberculous  and  non- 
tuberculous  guinea-pigs.  No  such  effects  had  ever  before  been  at- 
tained by  the  employment  of  any  therapeutic  agent.  It  is  small 
wonder  then  that  Koch  and  his  colaborers  were  surprised  at  the  re- 
sults observed,  and  readily  accepted  and  too  speedily  announced  the 
belief  that  a  specific  cure  for  tuberculosis  had  been  found.  The 
grounds  for  this  belief  were  strengthened  by  their  observation  of 
additional  evidence  in  the  selective  action  of  tuberculin. 

(2)  Not  only  does  tuberculin  select  tubercular  individuals  by  its 
action,  but  in  the  individual  it  selects  for  the  demonstration  of  its  most 
conspicuous  effects,  the  exact  site  of  the  tubercular  lesion.  If  a  man 
who  has  a  lupus  on  his  face  receives  a  tuberculin  injection  in  the 
back  or  in  any  other  portion  of  his  anatomy,  the  tissue  about  the 
lupus  soon  begins  to  show  evidence  of  stimulation  ;  it  becomes  hy- 
peremic,  the  margins  of  the  sore  begin  to  granulate,  and  if  the 
treatment  be  continued  the  lupus  often  temporarily  heals.  How- 
ever, more  extended  investigation  has  shown  that  the  action  of 
tuberculin  is  not  so  specific  as  was  at  first  believed.  The  experi- 
ments of  Krehl  have  demonstrated  the  fact  that  tubercular  animals 
are  especially  prone  to  show  elevations  of  temperature  after  the  in- 


SUPPURATION.  85 

jection  of  various  substances.  For  instance,  paralactic  acid  was 
found  to  act  similarly  to  tuberculin  and  on  section  of  tubercular 
animals  treated  with  this  agent  the  diseased  areas  were  observed  to 
be  hyperemic  and  to  contain  hemorrhagic  spots.  Matthes  ascertained 
that  certain  albumoses  and  peptons  affect  tubercular  animals  in  much 
the  same  way,  though  to  a  less  degree,  as  tuberculin  does. 

There  has  been  some  question  among  pathologists  concerning  the 
manner  in  which  tuberculin  acts  on  tubercular  tissue,  and  in  this 
connection  Baumgarten  makes  a  statement  which  may  be  condensed 
as  follows :  It  causes  an  exudative  inflammation  in  the  vascular 
tissue  about  the  tubercle,  and  in  this  way  the  tuberculous  tissue  may 
be  isolated  and,  when  situated  superficially,  removed.  In  some 
cases,  however,  after  the  prolonged  employment  of  the  agent,  the 
tuberculous  tissue  may,  under  the  influence  of  the  exudative  fluid 
and  the  polynuclear  leucocytes,  break  down  and  form  abscesses. 
The  bacilli  themselves  are  in  no  case  harmed  by  the  use  of  tuber- 
culin, and,  after  its  constant  employment  for  months,  they  retain  their 
original  form  and  lose  none  of  their  virulence.  Some  preparations 
seem  to  show  that  the  bacilli  multiply  more  rapidly  when  the  injec- 
tions are  made,  but  a  positive  statement  on  this  point  is  reserved  until 
further  studies  have  been  made.  It  is  certain,  however,  that  the 
non-tubercular  tissue  of  animals  acquires  no  immunity  against  the 
disease  from  the  injections.  This  is  shown  by  the  appearance  of 
metastatic  foci  in  animals  in  which  from  7  to  12  grams  of  the  orig- 
inal lymph  (an  amount  which  would  be  equivalent  to  from  70  to  180 
grams  in  man)  have  been  injected.  It  is  further  shown  by  the  fact 
that  in  some  animals  treated  subcutaneously  tubercles  have  appeared 
at  the  point  of  injection. 

Suppuration. — As  early  as  1879,  Leber  concluded  from  his  ob- 
servations on  infective  keratitis,  that  the  aspergillus  must  produce 
certain  soluble  products  which  diffuse  through  the  cornea  and  set  up 
an  inflammation  in  the  adjacent  vascular  tissue.  In  1882  he  showed 
that  suppuration  could  be  induced  by  the  introduction  of  sterilized 
mercury  and  copper,  and  that  the  pus  formed  is  free  from  micro5r- 
ganisms.  In  1884  he  induced  suppuration  by  the  injection  of  cul- 
tures of  the  staphylococcus  pyogenes  aureus  that  had  been  sterilized 
by  being  boiled  for  hours.  In  1888  he  reported  that  he  had  found 
an  alcoholic  extract  of  the  dried  staphylococcus  to  be  highly  pyoge- 
netic,  and  from  this  extract  he  prepared  a  crystalline  body  which  he 
calls  "  phlogosin."  This  substance  is  readily  soluble  in  alcohol  and 
ether,  sparingly  soluble  in  water,  and  crystallizes  in  needles.  The 
crystals  can  be  sublimed,  leaving  no  residue,  and  the  sublimate,  which 
forms  in  rosettes,  still  possesses  pyogenetic  properties.  Alkalis  pre- 
cipitate this  substance  from  its  solutions  in  amorphous  granules, 
which  dissolve  in  acids,  forming  crystalline  salts. 


86  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

Buchner  found  that  the  cells  of  many  bacteria  contain  pyogenetic 
proteids.  The  amount  of  these  substances  in  bacterial  cells  varies 
with  the  kind  of  germ,  and  some  species  (the  bacillus  prodigiosus, 
for  instance),  seem  to  contain  no  such  bodies.  The  bacillus  pyocy- 
aneus  contains  a  large  quantity  of  this  proteid  and  is  suitable  for 
lecture  demonstration.  The  germs  are  taken  from  potato  cultures, 
rubbed  up  with  water,  and  then  treated  with  about  fifty  volumes  of 
a  0.5  per  cent,  solution  of  caustic  potash.  This  forms  in  the  cold  a 
mucilaginous  mass  which  dissolves  at  the  temperature  of  the  water- 
bath,  and  after  being  heated  for  several  hours  the  fluid  is  filtered 
through  a  number  of  small  filters;  the  first  portion  should  be  refil- 
tered.  The  filtrate  is  a  greenish  fluid  (pyocyanin)  which  by  the 
careful  addition  of  acetic  or  hydrochloric  acid  (an  excess  is  to  be 
avoided)  forms  a  voluminous  precipitate  (pyocyaneus  proteid).  This 
precipitate  should  be  collected  on  a  filter,  washed  with  water,  then 
suspended  in  water,  and  a  few  drops  of  a  soda  solution  added,  when 
a  dark  brown  fluid,  with  a  tendency  to  gelatinize,  in  the  cold,  con- 
taining about  10  per  cent,  of  the  proteid,  is  obtained.  13.254  grams 
of  the  moist  bacteria  yield  1.44  gram  of  dry  bacterial  substance, 
and  this,  after  the  treatment  given  above,  furnishes  0.2739  gram  of 
dry  proteid  =  19.3  per  cent.  This  proteid  leaves  11.52  per  cent,  of 
ash,  which  contains  phosphoric  acid,  but  consists  principally  of  sodium 
chlorid.  Much  smaller  amounts  of  proteids  were  obtained  from 
other  organisms,  but  the  Eberth  germ,  bacillus  subtilis,  lactic  acid 
bacillus,  red  bacillus  from  potato,  and  staphylococcus  pyogenes 
aureus  furnish  considerable  quantities. 

The  chemotactic  properties  of  these  proteids  were  tested  in  the 
following  manner :  The  dissolved  proteid  was  placed  in  a  spindle- 
shaped  glass  tube,  and  this,  after  being  sterilized,  was  introduced 
under  the  skin  of  the  back  of  a  rabbit  with  antiseptic  precautions, 
and  the  ends  of  the  tube  broken  off  subcutaneously.  After  from 
two  to  three  days  the  tubes  thus  prepared  were  removed  and  found 
to  contain,  in  addition  to  some  of  the  proteid,  several  cubic  millime- 
ters of  fibrinous  pus,  which  was  examined  microscopically  and  by  the 
preparation  of  cultures,  which  remained  sterile.  The  proteid  of  the 
Eberth  bacillus  was  found  to  have  specially  marked  pyogenetic  prop- 
erties. More  extended  investigation  demonstrated  that  certain  other 
proteids  also  have  pyogenetic  properties.  The  subcutaneous  injec- 
tion of  sterilized  preparations  of  wheat  flour  and  ground  peas  caused 
suppuration.  Negative  results  were  obtained  with  starch  and  solu- 
tions of  di-sodium  hydric  phosphate ;  from  this  it  is  concluded  that 
the  active  agent  in  the  flour  is  its  gluten.  Pepton  was  employed 
without  effect,  while  gelatin  was  found  to  act  energetically.  Alka- 
line albuminates  were  prepared  from  muscle,  liver,  lungs  and  kidney, 
by  treating  finely  divided  portions  of  these  organs  with  potash  and 
proceeding  as  in  the  preparation  of  bacterial  proteids ;  all  of  these 


SUPPURATION.  87 

caused  the  formation  of  pus,  and  the  preparations  from  the  liver  were 
found  to  be  specially  potent.  Similar  preparations  from  blood  and 
egg  yolk  were  active,  while  those  from  fibrin  and  the  white  of  egg 
had  no  effect.  Hemi-albumose  was  also  found  to  be  active,  and  this 
fact  is  placed  in  contrast  with  the  negative  result  obtained  with 
pepton. 

The  bacterial  proteids,  also  some  of  the  vegetable  proteids,  when 
injected  directly  into  the  blood,  cause  a  general  leucocytosis.  A 
very  small  amount  of  the  proteid  of  the  pyocyaneus  injected  under 
the  skin  of  the  forearm  caused  the  following  symptoms  :  Two  hours 
after  the  injection  there  was  marked  pain  along  the  lymphatics,  es- 
pecially localized  in  the  elbow  and  axilla.  The  temperature  showed 
no  marked  elevation.  On  the  following  day  there  was  observed  a 
distinct  erysipelatous  redness  and  swelling  extending  for  some 
inches  about  the  place  of  injection,  and  this  was  accompanied  by 
severe  pain.  The  inflamed  area  felt  hard,  and  projected  distinctly 
above  the  surrounding  surface,  and  the  lymphatics  of  the  arm  ap- 
peared like  red  cords.  On  the  third  day  the  swelling  and  redness 
were  more  marked,  and  extended  from  the  wrist  to  the  elbow  ;  but 
on  the  fourth  day  the  symptoms  began  to  recede.  Here  we  have 
clinically  a  typical  erysipelas  with  lymphangitis,  and  Buchner 
claims  that  all  the  cardinal  symptoms  of  inflammation — rubor,  calor, 
dolor,  tumor — could  not  be  produced  without  involvement  of  the 
solid  tissues.  Similar,  but  less  marked,  symptoms  were  induced  by 
the  injection  of  a  dilute  solution  of  vegetable  casein. 

Buchner  states  that  bacteria  can  not  cause  inflammation  unless 
they  be  broken  down.  The  pyogenetic  substance  contained  within 
the  bacterial  cell  can  have  no  chemotactic  action  until  the  cell  disin- 
tegrates. Thus,  the  anthrax  bacillus  contains  a  pyogenetic  substance, 
but  no  pus  is  formed  in  mice  with  anthrax,  because  there  is  no  de- 
struction of  the  bacilli.  The  pyogenetic  proteid  of  the  anthrax 
bacillus,  however,  manifests  its  action  in  malignant  pustule. 

Many  non-pathogenic  germs  may  grow  in  wounds  and  by  elabor- 
ating their  poisons  may  increase  and  influence  the  general  intoxica- 
tion. Brunner  has  found  the  proteus  vulgaris  growing  in  a  wound, 
and  it  is  well  known  that  the  products  of  this  germ  are  powerful 
poisons. 

Mannotti,  after  treating  animals  with  sterilized  pus,  states  the 
following  conclusions  : 

(1)  Sterilized  pus  has  substantially  the  same  toxic  properties  as 
sterilized  cultures  of  the  staphylococcus.  (2)  Repeated  injections  of 
sterilized  pus  induce  chronic  intoxication  and  marasmus.  (3)  Injec- 
tion under  the  skin  causes  a  specially  grave  form  of  poisoning.  (4) 
The  symptoms  and  pathological  lesions  caused  by  these  injections 
correspond  with  those  observed  in  men  suffering  from  chronic  sup- 
puration. 


88  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

Van  de  Velde  ^  has  ascertained  that  the  staphylococcus  pyogenes 
produces  a  substance  which  destroys  leucocytes  and  for  which  the 
name  "  leukocidin  "  is  now  used.  He  injected  sterilized  cultures  of 
the  staphylococcus  into  the  pleural  cavities  of  rabbits  and  studied 
the  exudate  which  formed.  He  found  that  the  leucocytes  which  are 
present  in  such  an  exudate  in  large  numbers  soon  undergo  character- 
istic changes.  He  next  added  to  small  quantities  of  the  exudate 
living,  healthy  leucocytes  and  observed  that  these  underwent  a  like 
transformation.  Heating  the  exudate  for  ten  minutes  at  58°  pre- 
vents its  action  on  leucocytes.  The  successive  steps  in  the  destruc- 
tion of  the  white  blood  cells  are  as  follows  :  (1)  The  leucocytes  draw 
in  their  pseudopodia  and  become  round.  (2)  A  light  zone  appears 
in  the  periphery,  while  the  center  remains  granular.  (3)  The  light 
zone  gradually  extends  and  the  granulation  completely  disappears. 
(4)  The  corpuscles  are  apparently  reduced  to  empty  sacs  with  scarcely 
discernible  nuclei  which  finally  disappear  altogether.  These  degen- 
erative changes  are  complete  in  about  two  minutes.  Similar  changes 
are  observed  in  leucocytes  added  to  either  avirulent  or  virulent  cul- 
tures of  the  staphylococcus.  These  observations  have  been  con- 
firmed by  BaiP  and  Lingelsheim.^  Denys  and  Van  de  Velde* 
have  shown  that  the  serum  of  animals  rendered  immune  to  the 
staphylococcus  pyogenes  aureus  destroys  the  leukocidin  in  cul- 
tures of  this  germ.  Neisser  and  Wechsberg^  have  devised  a 
method  of  measuring  the  action  of  leukocidin  and  have  decided 
from  their  investigations  that  typical  pyogenetic  staphylococci,  both 
the  aureus  and  the  albus,  produce  the  same  leukocidin,  but  that 
there  are  varieties  of  both  these  organisms  which  do  not  produce 
this  poison. 

Krauss^  first  observed  that  many  red  blood  corpuscles  are  dis- 
solved by  filtered  cultures  of  the  staphylococcus.  This  hemolysin 
has  been  further  studied  by  Neisser  and  Wechsberg,  who  designated 
it  as  staphylolysin.  This  also  is  a  product  of  both  the  aureus  and 
the  albus.  It  is  believed  that  this  toxin  possesses  a  constitution 
similar  to  that  supposed  to  be  possessed  by  the  diphtheria  toxin  ac- 
cording to  Ehrlich's  theory,  and  that  it  consists  of  toxin  and  toxon. 
Furthermore,  it  is  probable  that  the  toxin  is  converted  into  toxoids ; 
but  while  the  toxoids  of  the  diphtheria  toxin  have  the  same  avidity 
for  antitoxin  as  is  possessed  by  the  toxin,  the  toxoids  of  staphylotoxin 
combine  with  antitoxin  with  less  avidity  than  do  the  toxins.  The 
blood  serum  of  healthy  men  contains  an  anti-staphylolysin,  but  that 
from  diflTerent  individuals  varies  greatly. 

'  La  cellule,  10. 

^Arcliivf.  Hyrfiene,  32. 

"  Aetiologie  unci  Tlierapie  der  Staphylokokken-Infectionen,  1900. 

*  La  cellule,  11. 

^  Zeilschrift  f.  Hygiene,  36. 

^  Wiener  klin.   Wochenschrift,  1900. 


SUPPURATION.  89 

Ribbert^  in  his  experiments  on  the  intravenous  injection  of  both 
sterilized  and  unsterilized  cultures  of  the  staphylococcus  in  rabbits 
observed  changes  in  the  kidneys,  heart,  lungs,  spleen,  and  bone  mar- 
row, which  he  attributed  to  the  action  of  the  toxin,  because  they 
were  found  after  the  injection  of  sterilized  as  well  as  after  the  use  of 
living  cultures.  These  changes  were  characterized  by  the  presence 
of  infarcts  and  abscesses.  Neisser  and  Wechsberg  observed  certain 
pathological  conditions  in  the  kidneys  of  animals  which  they  rendered 
immune  to  staphylotoxin  by  intravenous  injections.  Concerning 
these  kidney  lesions  they  make  the  following  statement :  They  are 
located  in  the  cortex ;  neither  the  parenchyma  nor  the  papillae  are 
involved.  In  the  cortex  one  readily  distinguishes  three  zones,  which, 
proceeding  from  the  periphery  towards  the  center,  are  as  follows  :  (1) 
A  circular,  irregular  zone  in  which  the  tubuli  contorti  are  for  the  most 
part  destroyed  and  the  peritubular  spaces  are  filled  with  fragments 
of  broken-down  leucocytes.  (2)  An  intermediate  zone  in  which  the 
leucocytic  reaction  is  less  marked,  but  in  which  necrosis  of  the  epi- 
thelium is  plainly  evident.  Only  a  small  part  of  the  epithelium  of 
the  tubuli  contorti  possess  nuclei  and  even  these  show  evidence  of 
cell  disintegration.  The  greater  part  of  the  cells  are  without  nuclei 
and  are  in  a  state  of  coagulation  necrosis.  The  uriniferous  tubules 
contain  hyaline  casts  and  fragments  of  broken-down  leucocytes.  In 
this  zone  the  glomeruli  are  markedly  altered.  One  observes  all 
changes  from  simple  hyperemia  to  complete  necrosis  in  which  there  is 
no  sign  of  a  glomerulus  left  except  a  structureless  capsule.  (3)  The 
third  zone,  like  the  first,  is  rich  in  broken-down  leucocytes,  espe- 
cially in  the  peritubular  spaces.  While  a  part  of  the  blood  vessels 
are  still  patent,  others  are  filled  with  thrombi  consisting  of  fibrin  and 
broken-down  leucocytes.  These  authors  were  unable  experimentally 
to  demonstrate  that  these  changes  in  the  kidney  are  directly  due  to 
the  staphylotoxin  and  they  have  provisionally  named  the  poison 
which  induces  these  lesions  "  nephrotoxin."  They  conclude  that  the 
hemolysin  and  the  leukocidin  of  the  staphylococcus  are  two  different 
poisons. 

The  action  of  sterilized  cultures  of  the  gonococcus  has  been  studied 
by  Christmas,^  Nikolaysen,^  and  Wassermann.*  It  has  been  found 
that  cultures  in  which  the  micro5rganism  has  been  destroyed  by  heat 
are  as  virulent  to  the  lower  animals  as  are  living  cultures.  Further- 
more, it  has  been  demonstrated  that  the  gonotoxin  is  contained  within 
the  cell  of  the  microorganism.  If  a  culture  two  days  old  be  filtered 
through  porcelain  and  the  filtrate  be  injected  into  mice,  but  little  or 
no  effect  results ;  while  if  a  culture  from  two  to  three  weeks  old  be 

1  Die  pathologische  Anatomie  und  die  Heilung  der  durch  dea  Staphjlococcua 
pyogenes  aureus  hervorgerufenen  Erkrankungen. 

^Annales  de  V  Institut  Pasteur,  1897. 
"  Centralblatt  f.  Bakteriologie,  1897. 
*  Zeitschrifi  /.  Hygiene,  1898,  27- 


90  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

treated  in  the  same  way  the  filtrate  is  found  to  be  toxic.  This  shows 
that  the  toxin  is  contained  within  the  cell  and  becomes  active  only 
after  the  disintegration  of  the  organism.  As  is  true  of  many  other 
bacteria,  different  cultures  vary  markedly  in  their  toxicity.  Wasser- 
mann  obtained  some  preparations,  0.1  c.c.  of  which  was  sufficient  to 
kill  mice,  while  one  c.c.  of  other  sterilized  cultures  was  necessary  in 
order  to  accomplish  the  same  result.  Gonotoxin  is  a  fairly  stable 
body  ;  it  can  be  precipitated  with  absolute  alcohol  and  can  be  heated 
to  100°  without  marked  loss  of  toxicity.  While  none  of  the  lower 
animals  are  susceptible  to  infection  with  this  micro(5rganism,  rabbits, 
mice  and  guinea-pigs  are  readily  affected  by  the  toxin.  The  sub- 
cutaneous injection  of  two  c.c.  of  a  sterilized  culture  of  a  virulent 
form  of  gonococcus  causes  an  infiltration  which  subsequently  passes 
into  a  necrosis.  Treatment  with  10  c.c.  causes  marked  local  inflam- 
mation, the  animal  loses  its  appetite,  wastes  away  and  dies  of  chronic 
marasmus.  The  pyogenic  action  of  this  toxin  can  easily  be  demon- 
strated by  the  injection  of  a  small  quantity  into  the  anterior  chamber 
of  the  eye  of  a  rabbit.  Corneal  cloudiness,  hypopyon  and  sometimes 
complete  loss  of  the  eye  result.  Wassermann  injected  0.1  c.c.  sub- 
cutaneously  into  his  own  arm.  After  about  four  hours  the  place  of 
injection  became  painful,  there  were  light  chills,  and  in  the  evening 
the  temperature  reached  38°.  During  the  night  there  was  headache 
and  pain  in  the  joints.  The  next  day  the  area  around  the  point  of 
injection  was  swollen  and  painful,  but  all  symptoms  disappeared 
after  two  days.  The  same  investigator  has  tried  this  agent  in  the 
treatment  of  obstinate  cases  of  chronic  gonorrhea,  but  has  found  that 
the  toxin  has  no  curative  effect  and  only  intensifies  the  symptoms. 
Both  he  and  Christmas  have  attempted  to  produce  an  antitoxin  for  this 
poison,  but  without  any  marked  success.  It  is  true  that  Christmas 
has  reported  the  preparation  of  an  active  serum  by  immunizing  goats, 
but  the  action  of  this  antitoxin  was  very  slight  and  Wassermann's 
results  were  wholly  negative.  Small  quantities  of  gonotoxin  intro- 
duced into  a  sound  urethra  cause  after  a  few  days  marked  suppura- 
tion, while  control  experiments  with  the  toxins  of  other  cocci  were 
without  effect.  The  action  of  gonotoxin  explains  the  clinical  course 
of  many  cases  of  gonorrhea.  When  the  infection  is  confined  to  the 
anterior  part  of  the  urethra  the  germ  soon  dies  and  the  toxin  is 
thoroughly  washed  out  with  each  discharge  of  urine ;  but  when  the 
infection  is  in  the  posterior  urethra  some  of  the  germs  find  their 
way  into  the  crypts  of  this  region  and  pass  through  many  genera- 
tions, elaborating  their  toxin  and  causing  the  continued  formation  of 
pus.  This  also  explains  why  this  disease  is  so  much  more  serious  in 
the  female  than  in  the  male. 

The  Summer  Diarrhoeas  of  Infancy. — In  1888,  Vaughan  stated 
that  the  microorganisms  which  produce  the  catarrhal  or  mucous  diar- 


THE  SUMMER  DIARRHCEAS  OF  INFANCY.  91 

rhcea  of  infancy,  are  probably  only  putrefactive  or  saprophytic  in 
character,  and  that  they  prove  harmful  by  producing  toxins ;  while 
those  that  cause  the  choleraic  form  or  serous  diarrhoeas,  are  more 
than  putrefactive,  they  are  pathogenic.  At  that  time  it  was  gener- 
ally believed  that  a  specific  germ  would  be  found  ;  but  the  truth  of 
the  above  statement  has  been  made  more  manifest  with  every  ex- 
perimental study  of  the  subject.  More  recently,  Booker '  makes  the 
following  statement :  "  No  single  microorganism  is  found  to  be  the 
specific  exciter  of  the  summer  diarrhoeas  of  infancy,  but  the  affection 
is  generally  to  be  attributed  to  the  result  of  the  activity  of  a 
number  of  varieties  of  bacteria,  some  of  which  belong  to  the  well- 
known  species  and  are  of  ordinary  occurrence  and  wide  distribution, 
the  most  important  being  the  streptococcus  and  proteus  vulgaris." 

Vaughan  has  studied  the  chemical  properties  of  the  germs  x,  a 
and  A  of  Booker's  list  in  the  following  manner  and  with  the  result 
as  stated  below  :  Beef  broth  cultures  were  kept  in  the  incubator  at 
37°  for  ten  days.  They  were  then  twice  filtered  through  heavy 
Swedish  filters,  and  the  second  filtrate  was  allowed  to  fall  into  a 
large  volume  of  absolute  alcohol,  feebly  acidified  with  acetic  acid. 
A  voluminous,  flocculent  precipitate  resulted  in  each  case,  and  after 
subsidence  the  supernatant  fluid  was  decanted.  The  precipitates 
were  then  treated  with  distilled  water,  in  which  those  from  x  and  a 
were  soluble,  while  that  from  A  proved  insoluble.  A  large  volume 
of  absolute  alcohol  was  again  added,  and  the  mixture  allowed  to 
stand  for  four  days.  The  precipitates  from  x  and  a  completely 
subsided,  leaving  the  supernatant  fluids  perfectly  clear ;  but  in  the 
case  of  A  the  subsidence  was  not  complete.  The  precipitates  were 
collected,  by  decantation  and  filtration,  on  porous  plates,  and  dried 
over  sulphuric  acid.  These  substances  are  proteid  in  composition 
but  differ  from  known  proteids  and  from  one  another.  That  from  x 
is  slightly  yellow,  as  seen  deposited  in  the  alcohol,  but  becomes 
grayish  on  exposure  to  the  air.  It  is  readily  soluble  in  water  from 
which  it  is  precipitated  by  either  heat  or  nitric  acid,  singly  or  combined. 
It  gives  the  biuret  and  xanthoproteic  reactions  and  is  precipitated 
by  saturating  its  aqueous  solution  with  ammonium  sulphate,  and 
therefore  cannot  be  classed  with  the  peptons.  Sodium  sulphate  and 
carbonic  acid  fail  to  throw  it  down  from  its  aqueous  solution  ;  conse- 
quently it  is  not  a  globulin,  and  for  the  present  at  least  it  must  be 
classified  among  the  albumins ;  however,  it  possesses  properties  which 
do  not  belong  to  the  known  albumins. 

The  proteid  prepared  from  cultures  of  the  germ  a  is,  as  seen  under 
the  alcohol,  very  light,  flocculent,  and  perfectly  white  ;  but  so  soon 
as  it  is  brought  in  contact  with  the  air  it  begins  to  blacken,  and 
finally  dries  down  on  the  porous  plate  in  black  scales.  It  possesses 
the  same  general  properties  in  regard  to  the  action  of  solvents  and 
^  Johns  Hopkins  Hospital  Reports,  6,  1897. 


92  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

other  reagents  which  were  found  to  be  possessed  by  the  proteid  ob- 
tained from  cultures  of  x. 

The  proteid  of  J.  is  peculiar,  inasmuch  as  it  is  practically  insoluble 
in  water. 

All  these  proteids  are  highly  poisonous,  and  when  injected  under 
the  skin  of  kittens  or  dogs  cause  vomiting  and  purging,  and,  when 
employed  in  sufficient  quantity,  collapse  and  death.  Post-mortem 
examination  shows  the  small  intestine  pale  throughout  and  con- 
stricted in  places.  The  heart  is  found  to  be  in  diastole  and  filled 
with  blood.  The  following  brief  notes  from  the  record  of  experi- 
ments illustrate  the  nature  of  the  symptoms  and  the  post-mortem 
appearances  : 

A  small  amount  of  proteid  from  bacillus  x,  dissolved  in  water, 
was  injected  under  the  skin  on  the  back  of  a  kitten  about  eight 
weeks  old.  Within  one-half  hour  the  animal  began  to  vomit  and 
purge,  and  death  resulted  within  eighteen  hours.  The  small  intes- 
tines were  pale,  contracted  in  places,  and  contained  a  frothy  mucus. 
The  stomach  was  distended  with  gas  and  contained  yellowish  mucus. 
The  liver  appeared  normal,  the  spleen  and  the  kidneys  congested, 
and  the  heart  distended.  , 

Another  kitten  was  treated  with  a  proteid  from  bacillus  a  dis- 
solved in  water.  The  vomited  and  fsecal  matters  in  this  case  were 
green.  The  animal  died  after  fifteen  hours,  and  presented  appear- 
ances practically  identical  with  those  mentioned  above. 

A  third  kitten  was  treated  with  some  of  the  proteid  of  bacillus  A, 
suspended  in  water,  and  presented  substantially  the  same  symptoms 
and  post-mortem  appearances. 

A  fourth  animal  was  treated  in  the  same  manner  as  above  with 
the  proteid  prepared  from  some  canned  meat.  This  was  done  as 
a  control  on  the  above  experiment,  and  the  kitten  remained  unaf- 
fected, thus  demonstrating  the  fact  that  the  poisonous  properties 
are  peculiar  to  the  bacterial  proteids. 

Concerning  the  amount  of  these  proteids  necessary  to  produce  fatal 
results  in  the  animals  experimented  upon,  the  following  statement  may 
be  made  :  Under  the  skin  on  the  back  of  a  guinea-pig  10  mg.  of  the 
dry  scale  proteid  from  bacillus  a  was  injected,  and  caused  death 
within  twelve  hours.  Of  two  kittens  treated  with  15  mg.  each  of 
the  a-albumin,  one  died  after  forty-eight  hours,  and  the  other  re- 
covered after  two  days  of  purging  and  vomiting.  Two  dogs,  of 
about  five  pounds'  weight,  had  each  40  mg.,  and  after  serious  illness 
of  two  days'  duration,  recovered.  During  these  two  days  of  purging 
and  vomiting  the  dogs  were  constantly  shivering  as  with  cold,  but 
the  rectal  temperature  stood  at  from  102.5°  to  103.5°  F.  There 
was  in  no  case  any  sign  of  inflammation  at  the  point  of  injection. 
Plate  cultures  were  made  from  the  proteids  themselves,  and  from  the 
blood,  liver,  spleen,  and  kidneys  of  some  of  the  animals  killed  with 


THE  SUMMER  DIARRHCEAS  OF  INFANCY.  93 

the  proteid,  and  these  remained  sterile,  thus  demonstrating  that  no 
germ  was  introduced  into  the  animals  along  with  the  chemical 
poison. 

The  following  conclusions  concerning  the  germs  which  cause  the 
summer  diarrhoeas  of  infancy  may  be  formulated  : 

1.  There  are  many  microorganisms,  any  one  of  which,  when  intro- 
duced into  the  intestines  of  the  infant,  under  certain  favorable  con- 
ditions may  produce  diarrhoea. 

2.  Many  of  these  microorganisms  are  true  saprophytes.  A  germ 
growing  in  the  intestine  does  not  necessarily  feed  upon  living  tissue. 
The  food  in  the  duodenum  before  absorption  has  no  more  vitality  than 
the  same  material  in  a  culture  flask.  Moreover,  the  excretions 
poured  into  the  intestines  from  the  body  are  not  possessed  of  vitality. 
A  bacterium  which  will  grow  upon  a  certain  medium  in  a  flask  and 
produce  a  poison  will  grow  on  the  same  medium  in  the  intestine  and 
produce  the  same  poison,  provided  it  is  not  destroyed  by  some  secre- 
tion of  the  body. 

3.  The  only  digestive  secretion  which  is  known  to  have  any  de- 
cided germicidal  effect  is  the  gastric  juice  ;  therefore,  if  this  secretion 
be  impaired  there  is  at  least  the  possibility  that  the  living  germ  will 
pass  on  to  the  intestine,  will  there  multiply,  and  will,  if  it  be  capable 
of  so  doing,  elaborate  a  chemical  poison  which  may  be  absorbed.  It 
has  been  said  that  the  gastric  juice  does  not  act  as  a  germicidal 
agent,  because  there  are  other  acids  which  are  more  powerfully  bac- 
tericidal than  hydrochloric  acid,  but  there  is  no  force  in  this  argu- 
ment. The  question  is  not  whether  the  stomach  is  supplied  with  the 
very  best  germicide,  but  whether  it  is  supplied  with  any  at  all.  The 
human  eye  is  not  by  any  means  a  perfect  mechanism,  but  it  is  man's 
only  organ  of  vision. 

The  chief  reason  why  the  breast-fed  child  has  a  better  chance  for 
life  than  the  one  fed  upon  cow's  milk  lies  in  the  fact  that  the  former 
gets  its  food  germ-free ;  but  a  second  reason  is  to  be  found  in  the 
large  amount  of  acid  required  to  neutralize  the  cow's  milk,  as  has 
been  pointed  out  by  Escherich.  It  is  also  possible  that  some  of  the 
secretions  poured  into  the  intestines  have  germicidal  properties,  or 
that  the  cells,  in  absorbing  the  bacterial  proteids,  may  to  a  limited 
extent  so  alter  them  that  they  are  no  longer  poisonous,  or  that  in  a 
perfectly  normal  condition  the  liver  may  be  able  to  prevent  these 
poisons  from  entering  the  general  circulation  without  change. 

4.  Any  germ  which  is  capable  of  growing  and  producing  an  ab- 
sorbable poison  in  the  intestines  may,  for  all  practical  purposes,  be 
considered  pathogenic.  It  is  not  necessary  that  a  bacterium  be  cap- 
able of  growing  and  causing  disease  and  death  when  injected  under 
the  skin  or  into  the  blood  in  order  to  establish  its  right  to  rank 
with  the  pathogenic  germs.  In  the  blood  the  organism  is  acted 
upon  by  a  wholly  different  fluid  from  that  by  which  it  is  surrounded 


94  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

in  the  intestines,  and  the  germicidal  properties  of  the  blood  have 
been  unquestionably  demonstrated. 

5.  The  proper  classification  of  bacteria  in  regard  to  their  relation 
to  disease  can  not  be  made  from  their  morphology  alone,  but  must 
depend  largely  upon  the  products  of  their  growth.  As  has  been 
stated,  three  microorganisms,  differing  sufficiently  to  be  recognized 
as  of  different  species,  produce  poisons,  all  of  which  induce  vomiting 
and  purging,  and  when  used  in  sufficient  quantity,  death.  Morpho- 
logically these  bacilli  may  not  be  closely  related,  but  physiologically 
they  are  near  akin.  If  these  deductions  be  true,  we  will  avoid  the 
introduction  into  the  alimentary  canal,  not  only  of  the  specific  patho- 
genic germs,  but  of  all  toxicogenic  microorganisms. 

Baginsky  and  Stadthagen  obtained  from  cultures  of  the  "  white 
liquefying  bacterium  "  of  the  former  a  poisonous  proteid  which  pro- 
duces in  mice,  after  about  five  hours,  slight  dyspnoea,  the  coat  be- 
comes rough,  the  animal  sits  with  drooping  head,  and  when  forced 
to  move  does  so  sluggishly  but  without  any  evidence  of  paralysis. 
The  marked  apathy  increases,  and  death  results  after  two  or  three 
days.  Section  shows  an  infiltration  about  the  place  of  injection, 
congestion  of  the  spleen,  liver  and  peritoneum.  The  intestine  is 
hyperemic  throughout  its  entire  length,  and  its  upper  portion  con- 
tains a  reddish-brown  fluid.  They  also  obtained  from  the  same 
cultures  a  poisonous  ptomam,  which  is  probably  identical  with 
one  found  by  Brieger  in  putrid  horse  flesh,  and  has  the   formula 

That  tyrotoxicon  is  one  of  the  causes  of  the  violent  choleraic  diar- 
rhoea of  children  there  can  scarcely  be  a  doubt.  The  symptoms  in- 
duced by  the  poison  cannot  be  distinguished  from  those  induced  by 
the  disease.  The  post-mortem  appearances  are  very  much  alike,  it 
not  identical,  and  the  poison  has  been  found  in  milk,  a  part  of  which 
had  been  given  to  a  child  not  more  than  two  hours  before  the  first 
symptom  of  a  violent  attack  of  the  disease  made  itself  manifest ;  but 
tyrotoxicon  is  not  so  frequently  found  in  milk  as  was  at  one  time 
supposed. 

Fliigge  has  studied  milk  bacteria  with  special  reference  to  their 
toxicogenic  properties.  In  market  milk  he  has  frequently  found 
four  anaerobic  bacilli,  two  of  which  produce  poisons.  The  subcuta- 
neous injection  of  filtered  cultures  of  one  of  these  in  doses  of  from 
0.3  to  0.6  c.c.  in  mice  caused  death  after  from  three  to  fifteen  hours, 
and  section  showed  marked  hyperemia  of  the  intestines  and  trans- 
udates in  the  peritoneal  and  pleural  cavities.  The  intra-abdominal 
injection  of  five  c.c.  of  this  culture  killed  guinea-pigs  within  from 
fifteen  to  twenty-four  hours,  and  in  these  also  the  intestines  were 
found  to  be  engorged  and  the  abdominal  cavity  filled  with  a  serous 
transudate.  The  second  toxicogenic,  anaerobic  bacillus  develops  a 
most  disagreeable  odor  in  milk,  and  consequently  it  is  not  likely 


THE  SUMMER  DIARRHCEAS  OF  INFANCY.  96 

to  be  taken.  Summer  diarrhoeas  can  scarcely  be  attributed  to  these 
anaerobic  germs,  and  yet  they  cannot  be  regarded  as  altogether 
harmless. 

Fliigge  found  and  isolated  twelve  species  of  peptonizing  bacteria 
in  market  milk  and  three  of  these  were  found  to  be  markedly  poison- 
ous to  animals.  Concerning  the  action  of  cultures  of  these  bacilli, 
the  following  statement  is  made  :  "  The  two  days'  old  cultures  of 
No.  I.  induce  in  frogs,  on  the  injection  of  two  c.c.  into  the  dorsal  lymph 
zone,  first  slowness  of  motion  and  reflexes,  after  one  hour  paralysis  of 
the  extremities  and  complete  loss  of  reaction,  and  after  four  hours, 
death.  Mice  die  after  from  five  to  six  hours  from  the  subcutaneous 
injection  of  0.5  c.c.  With  the  exception  of  lack  of  voluntary 
motion  and  tardiness  of  reaction,  no  symptoms  are  manifest. 
Guinea-pigs,  that  have  received  five  c.c.  intra-abdominally,  lie  on  the 
side  and  have  marked  dyspnoea,  the  abdomen  is  retracted,  and  hand- 
ling them  causes  pain.  Death  results  after  from  four  to  seven  hours. 
Section  shows  hyperemia  of  the  kidneys,  and  the  peritoneal  and 
serous  coat  of  the  intestines  are  markedly  reddened.  Nothing  else 
of  interest  in  found.  Dogs  drink  the  milk  cultures  with  relish  and 
in  large  quantities.  After  one  hour  severe  diarrhoea  sets  in  with  a 
movement  every  five  minutes.  When  fed  with  normal  milk  recov- 
ery follows. 

"  Two  days'  old  milk  cultures  of  No.  III.  induce  in  frogs  and  mice 
no  symptoms.  Guinea-pigs  and  rabbits  receiving  intra-abdominal  or 
intravenous  injections  remain  quiet  in  their  cages,  respond  quickly 
to  irritation,  but  gradually  recover.  The  cultures,  when  fed  to 
puppies,  induce  sharp  diarrhoea  and  apparently  severe  pain  in  the 
abdomen.  One  of  the  puppies  showed  on  the  second  day  progressive 
exhaustion,  paralytic  weakness  of  the  extremities,  and  a  fall  of  tem- 
perature. He  died  on  the  third  day,  and  section  showed  hyperemia 
of  the  kidneys,  nothing  else  worthy  of  note." 

"  Bacillus  No.  VII.  injected  in  milk  cultures  into  frogs,  mice  and 
guinea-pigs  had  no  marked  action.  When  the  culture  was  filtered 
through  a  Chamberland  filter  and  concentrated  in  vacuo  to  one-fifth 
its  volume,  it  killed  mice  and  guinea-pigs  when  injected  in  doses  ot 
0.6  and  5  c.c.  respectively.  Death,  which  followed  in  from  six  to 
twelve  hours,  was  preceded  by  dyspnoea  and  convulsive  movements. 
Section  showed  nothing  characteristic.  Even  the  unconcentrated 
milk  cultures  acted  powerfully  when  fed  to  puppies.  After  feeding 
for  one  or  two  days,  profuse  diarrhoea  set  in,  but  disappeared  the 
next  day.  The  diarrhoea  was  accompanied  by  great  emaciation, 
weakness  of  the  extremities,  and  tottering  gait.  As  soon  as  the 
use  of  the  cultures  was  discontinued,  and  ordinary  milk  given,  im- 
provement began  and  continued  to  complete  recovery.  Two  puppies, 
after  recovery,  were  again  fed  with  the  cultures,  and  after  a  short  time 
the  profuse  diarrhoea  with  its  accompanying  symptoms  reappeared." 


96  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

Fliigge  discussed  the  question  as  to  whether  or  not  the  poisonous 
properties  of  these  peptonizing  bacilli  are  due  to  the  peptons  formed, 
and  came  to  the  conclusion  that  this  could  not  be  the  case.  He 
found  among  the  twelve  species  some  that  produced  peptons  much 
more  energetically  than  do  the  harmful  kinds.  In  fact,  one  of  the 
most  highly  virulent  species  produced  the  least  pepton.  Further- 
more, the  symptoms  that  followed  the  injections  were  not  those  that 
are  induced  by  pepton.  It  is  true  that  different  investigators,  who 
have  tested  the  nutritive  value  of  peptons  and  albumoses  on  the 
lower  animals  and  on  healthy  and  sick  men,  are  unanimous  in  the 
verdict  that  the  long-continued  employment  of  these  preparations 
causes  in  both  men  and  dogs  severe  intestinal  irritation.  Ziintz 
noted  that  dogs  fed  on  peptons  suffered  from  an  abundant,  watery 
diarrhcea,  and  eliminated  from  three  to  six  times  as  much  nitrogen 
unused  as  those  fed  upon  meat,  and  Munk  obtained  like  results. 
Pfeiffer  induced  in  himself  and  in  another  man  intestinal  irritation 
and  diarrhoea  by  large  doses  of  pepton  and  Neumeister  states  :  "  By 
long-continued  use  of  these  preparations,  symptoms  of  marked  irri- 
tation and  injury  to  the  intestines  uniformly  resulted,  and  con- 
sequently the  prescription  of  albumoses  in  disease  can  scarcely  be 
regarded  as  '  rational.' "  The  reason  why  partly  digested  milk  is 
often  so  well  borne  by  children  is  due  to  the  fact  that  but  little 
pepton  is  formed,  and  from  all  that  we  know  of  the  nutritive  value 
of  albumoses  and  peptons,  the  long-continued  employment  of  pep- 
tonized milk  in  the  feeding  of  infants  cannot  be  recommended ;  but 
Fliigge  was  right  in  concluding  that  the  toxic  properties  of  cultures 
of  peptonizing  bacteria  found  in  milk  by  him  were  not  due  to  the 
peptons.  This  has  been  conclusively  shown  by  more  recent  re- 
searches in  Fliigge's  laboratory  prosecuted  by  Liibbert.^  This  in- 
vestigator has  extended  the  study  of  one  of  the  most  virulent  species 
of  peptonizing  bacilli  found  by  Fliigge.  He  has  shown  that  this 
microorganism  rapidly  digests  the  casein  of  milk,  while  it  has  no 
action  on  either  the  fat  or  the  lactose.  Pure  milk  cultures  of  this 
bacillus  were  readily  taken  by  guinea-pigs  and  caused  death  within 
four  days.  Three  young  dogs  fed  with  such  cultures  developed 
severe  diarrhoea  after  two  hours  and  died  within  from  four  to  seven 
days ;  while  older  dogs  consumed  large  quantities  of  the  milk  and 
remained  unaffected.  This  is  especially  interesting,  inasmuch  as  it 
is  a  well-known  fact  that  after  the  second  year  of  life,  children  sel- 
dom suffer  from  summer  diarrhoeas.  Section  of  the  animals  killed 
with  these  cultures  showed  slight  swelling  and  injection  of  the 
mucous  membrane  of  the  intestine.  The  bacteria  could  not  be  dis- 
covered outside  of  the  intestines,  either  in  the  blood  or  the  organs. 
This  demonstrates  that  death  resulted  from  intoxication  and  was  not 
due  to  sepsis.  Further  researches  demonstrated  the  fact  that  the 
'  Zeitschriftf.  Hygiene,  22,  1. 


TYPHOID  FEVER.  97 

toxin  is  contained  within  the  bacterial  cells  and  when  all  these  are 
removed  by  filtration,  the  filtrate  is  without  harmful  effect.  These 
investigations  indicate  that  the  toxin  is  absorbed  from  the  alimentary 
canal  of  young  animals,  while  it  is  not  taken  up  from  the  digestive 
tract  of  adults.  Both  the  gastric  and  pancreatic  juices  were  found 
to  be  without  effect  upon  these  bacilli,  and  the  filtrates  obtained  after 
digestion  of  the  bacterial  cells  in  these  fluids  were  without  poisonous 
action.  Inasmuch  as  no  bacteria  were  found  in  the  blood  of  any  of 
the  internal  organs,  we  must  conclude  that  the  germ  cells  undergo  a 
process  of  disintegration  or  digestion,  while  being  absorbed  through 
the  intestinal  walls.  Boiling  milk  cultures  of  this  toxicogenic,  pep- 
tonizing bacillus  destroys  their  virulence  but  it  must  not  be  inferred 
from  this  that  all  milk  containing  toxicogenic  germs  is  rendered 
harmless  by  heat.  The  colon  bacillus  is  frequently  found  in  market 
milk  and  it  is  altogether  likely  that  this  germ  plays  an  important 
role  in  the  causation  of  the  summer  diarrhoeas  of  infancy  and,  as 
we  shall  see  later,  the  colon  toxin  resists  very  high  temperatures. 

Typhoid  Fever. — The  specific  character  of  the  bacillus  discovered 
by  Eberth  in  1880  has  been  abundantly  demonstrated  by  subsequent 
bacteriological  research,  and  there  is  no  doubt  that  this  microorgan- 
ism is  the  cause  of  typhoid  fever.  In  1885,  Brieger  obtained  from 
pure  cultures  of  the  Eberth  bacillus  a  poisonous  ptomain,  which  pro- 
duced in  guinea-pigs  a  slight  flow  of  saliva,  frequency  of  respiration, 
dilatation  of  the  pupils,  profuse  diarrhoea,  paralysis,  and  death  within 
from  twenty-four  to  forty-eight  hours.  Post  mortem  examination 
showed  the  heart  in  systole,  the  lungs  hyperemic,  and  the  intestines 
contracted  and  pale.  At  first  Brieger  was  inclined  to  regard  this  as 
the  specific  poison  of  typhoid  fever,  and  named  it  typhotoxin,  but 
more  extended  investigation  has  shown  that  this  substance  cannot  be 
regarded  as  the  essential  toxin  of  the  Eberth  bacillus.  In  their  re- 
searches upon  the  toxalbumins,  Brieger  and  Frankel  found  in  cul- 
tures of  the  typhoid  bacillus  a  proteid  which  caused  death  in  rabbits 
after  from  eight  to  ten  days.  This  substance  undoubtedly  contained 
the  typhoid  toxin  but  was  by  no  means  a  pure  preparation  of  this 
substance. 

Pfeiffer  has  demonstrated  that  typhotoxin  is  contained  in  the  bac- 
terial cells  and  he  has  shown  that  4  mg.  of  the  dried  cells  for  each 
100  grams  of  body  weight  suffice  to  kill  guinea-pigs.  With  this 
poison  animals  can  be  rendered  immune  to  the  Eberth  bacillus,  and 
the  blood  of  such  immune  animals  contains  a  slightly  active  anti- 
toxin, which,  however,  has  not  proved  of  value  in  the  treatment  of 
the  disease. 

In  1889,  Vaughan  isolated  from  mixed  cultures  from  typhoid 
stools  a  base,  forming  crystalline  salts  and  capable  of  inducing  in 
cats  and  dogs  a  marked  elevation  of  temperature  accompanied  by 
7 


98  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

severe  purging.  The  following  is  the  record  of  one  experiment  with 
this  substance  :  "  An  aqueous  solution  of  the  crystals  was  given  to  a 
dog  by  the  mouth  at  3  p.  m.  The  rectal  temperature  was  101"  F. 
before  the  administration.  At  3.15  retching  and  vomiting  set  in  and 
continued  at  intervals  for  more  than  two  hours.  At  3.30  the  tem- 
perature was  103°  F.  and  at  3.55  the  animal  began  to  purge.  The 
first  discharges  contained  much  fecal  matter,  but  subsequently  they 
were  watery  and  contained  mucus  stained  with  blood.  At  4,  the 
temperature  was  103.5°  F.,  and  remained  the  same  at  4.30.  The 
animal  was  seen  again  at  10  a.  m.,  the  next  day,  when  its  temper- 
ature was  101.5°  and  recovery  seemed  complete." 

This  base  was  not  obtained  in  sufficient  quantities  for  an  ultimate 
analysis.  The  platino-chlorid  crystallizes  in  fine  rhombic  prisms 
and  the  hydrochlorid  in  long,  delicate,  red  needles.  The  red  color 
seems  to  be  inherent  to  the  substance  and  not  due  to  impurities.  The 
mercury  and  platinum  compounds  are  insoluble  in  alcohol,  soluble  in 
water.     The  hydrochlorid  is  soluble  in  both  water  and  alcohol. 

In  1890,  Vaughan  reported  the  isolation,  from  water  supposed  to 
cause  typhoid  fever,  of  a  number  of  toxicogenic  germs,  and  the 
chemical  properties  of  two  of  these  have  been  studied.  They  belong 
to  the  proteids,  and  an  analysis  of  one  of  these  by  Freer  shows  it  to 
belong  to  the  nucleins.  These  poisons  are  soluble  in  water,  the 
opalescent  solution  showing  a  distinctly  acid  reaction.  They  are  not 
precipitated  by  heat  or  nitric  acid  singly  or  combined.  They  dissolve 
in  nitric  acid,  forming  a  colorless  solution,  which  becomes  yellow  on 
the  addition  of  ammonia.  They  dissolve  in  caustic  alkalis,  and  the 
solution  becomes  purple  on  the  addition  of  copper  sulphate. 

On  white  rats  these  poisons  produced  symptoms  which  are  identi- 
cal with  those  following  inoculations  with  the  living  germs.  The  rat 
seems  to  shiver  with  cold  and  gives  evidence  of  abdominal  pain. 
It  lies  with  its  limbs  flexed  and  head  drawn  down  for  a  few  seconds, 
then  stretches  out  the  limbs.  It  lies  on  the  side  for  a  short  time,  then 
sits  with  the  head  drawn  under  the  body.  Dogs  shiver  as  with  cold, 
but  at  the  same  time  the  rectal  temperature  is  from  1°  to  4°  above 
the  normal.  The  following  experiments  seem  to  show  that  the 
poison  accumulates  in  the  nerve  centers  :  Two  guinea-pigs  were 
treated  with  hypodermic  injections  of  one  of  these  poisons,  the 
amount  used  being  about  ten  times  the  dose  which  ordinarily  proves 
fatal  to  one  of  these  animals.  Within  twelve  hours  both  were  dead. 
Plate  cultures  made  from  the  liver,  blood,  spleen,  brain  and  spinal 
cord  remained  sterile.  Small  quantities  of  the  brain  and  spinal 
cord  were  rubbed  up  in  a  sterilized  dish  with  sterilized  water,  and 
two  c.c.  of  the  emulsion  was  injected  under  the  skin  of  each  of 
four  guinea-pigs.  These  animals  seemed  to  be  very  excitable  the 
next  day,  throwing  themselves  about  violently  in  their  cages  when 
slight  noises  were  made  near  them.     Within  a  period  of  from  six- 


HOG   CHOLERA.  99 

teen  to  twenty-four  days  all  died.  This  experiment  needs  repetition 
and  it  will  be  necessary  to  prepare  and  inject  similar  emulsions  made 
from  other  organs  before  any  positive  conclusions  can  be  drawn. 

Hog  Cholera. — In  pure  cultures  of  this  bacillus  Novy  has  found 
a  poisonous  base,  which  probably  has  the  composition  Cj^Hj^Nj,  and 
to  which  he  has  provisionally  given  the  name  susotoxin.  100  mg. 
of  the  hydrochlorid  of  this  base  causes  in  white  rats  convulsive  tre- 
mors and  death  within  one  and  one-half  hours.  Section  shows  the 
heart  in  diastole,  lungs  pale,  stomach  contracted,  a  serous  effusion 
into  the  thoracic  cavity,  and  the  subcutaneous  tissue  pale  and  edem- 
atous. 

Novy  has  also  obtained  a  poisonous  proteid  from  cultures  of  this 
germ.  The  following  experiments  illustrate  the  effects  obtained  from 
this  body  :  100,  50  and  25  mg.,  respectively,  were  injected  into  three 
young  white  rats  from  the  same  litter.  The  animal  which  received 
100  mg.  soon  began  to  crawl  about  on  its  belly,  being  unable  to  rise. 
The  eyes  were  soon  filled  with  a  thick  secretion  and  the  toes  became 
red.  Finally  it  became  quiet,  lying  on  its  belly,  with  feet  extended. 
The  respirations  became  deeper,  and  a  coma-like  condition  set  in. 
The  animal  died,  without  convulsions,  within  about  three  hours. 
The  rat  which  received  50  mg.,  went  through  the  same  course  of 
symptoms,  but  these  were  less  intense.  Death  resulted  four  hours 
after  the  injection.  The  one  which  received  the  25  mg.  became  very 
sick,  but  finally  recovered,  and  one  week  later  it  was  given  another 
injection  of  30  mg.,  which  produced  scarcely  any  effect.  Then  it 
was  treated  at  intervals  of  five,  three,  five,  two  and  four  days,  re- 
spectively, to  40,  50,  75,  100  and  125  mg.  without  effect.  Three 
days  after  the  last  injection  the  animal  was  inoculated  with  one  c.c. 
of  a  bouillon  culture  of  the  highly  virulent  germ.  Only  a  slight 
temporary  effect  was  observed  during  the  first  day,  after  which  re- 
covery was  complete  and  permanent.  A  control  rat  was  given  the 
same  quantity  of  the  culture,  sickened  the  next  day  and  died  one 
week  later.  From  this  it  will  be  seen  that  the  animal  was  rendered 
immune  to  the  bacillus. 

De  Schweinitz  also  reports  the  detection  of  a  slightly  poisonous 
base,  which  he  designated  sucholotoxin,  and  a  poisonous  proteid,  and 
with  these  he  has  been  able  to  secure  immunity  in  guinea-pigs  against 
the  virulent  germ.  The  proteid  body  is  classed  among  the  albumoses, 
and  is  said  to  crystallize  in  white,  translucent  plates  when  dried  in 
vacuo  over  sulphuric  acid  and  to  form  needle-like  crystals  with 
platinum  chlorid.  The  same  investigator  has  also  reported  the  iso- 
lation of  soluble  ferments,  or  enzymes,  from  cultures  of  the  hog 
cholera  bacillus.  From  milk  cultures  both  peptonizing  and  diastatic 
ferments  were  obtained.  These  are  destroyed  by  heating  above  55"  ; 
they  contain  nitrogen,  and,  when  pure,  do  not  give  the  albuminoid 


100  BACTERIAL  POISONS  OF  INFECTIOUS  DISEASES. 

reactions.  Injections  of  the  soluble  ferments  conferred  immunity. 
In  1892  Novy  obtained  immunity  in  rabbits  by  injection  of  blood 
serum  of  immunized  animals,  and  similar  results  were  obtained 
by  De  Schweinitz.  In  the  same  year  Metschnikoff  published 
his  studies  on  immunization  with  blood  serum  of  rabbits  im- 
mune to  hog  cholera,  but  according  to  Smith  and  Moore,  he  did 
not  work  with  the  hog  cliolera  germ,  but  with  that  of  swine 
plague.  The  last-mentioned  individual  obtained  only  partial  im- 
munity with  blood  serum  of  immune  guinea-pigs,  and  none  with 
that  of  rabbits. 

Rabbit  Septicemia. — Hoffa  has  killed  rabbits  by  inoculation  with 
pure  cultures  of  the  bacillus  of  this  disease,  and  has  isolated  from 
the  bodies  of  these  animals  methylguanidin,  while  in  the  bodies  of 
healthy  rabbits  this  poison  could  not  be  found.  He  ascertained  that 
the  average  fatal  dose  of  methylguanidin  for  rabbits  is  0.2  gm. 
when  given  subcutaneously.  Hueppe  thinks  that  the  bacterium  of 
chicken  cholera  is  identical  with  that  of  rabbit  septicaemia,  and  it 
has  been  ascertained  that  chickens  also  may  be  poisoned  with 
methylguanidin  and  that  the  symptoms  induced  by  this  substance 
resemble  those  of  the  disease. 

Pneumonia. — Bonardi  reported  the  presence  of  certain  poisonous 
basic  substances  in  cultures  of  the  diplococcus  of  Frankel,  but  he 
was  unable  to  obtain  these  in  quantity  sufficient  for  ultimate  analysis. 
He  also  stated  that  he  secured  immunity  against  the  germ  by  treat- 
ing rabbits  with  a  small  quantity  of  the  chemical  poisons.  How- 
ever, temporary  immunity  to  the  diplococcus  may  be  induced  by 
non-specific  agents,  and  Vaughan  ^  concluded  a  research  upon  this 
subject  as  follows  : 

1.  Rabbits  and  guinea-pigs  maybe  protected  against  virulent  cul- 
tures of  the  diplococcus  of  pneumonia  by  previous  treatment  with 
hypodermatic  injections  of  a  solution  of  yeast  nuclein. 

2.  The  immunity  thus  secured  is  not  due  to  the  action  of  the 
nuclein,  as  a  germicide,  directly  on  the  germ. 

3.  The  process  of  securing  this  immunity  is  an  educational  one, 
and,  most  probably,  depends  upon  the  stimulant  efPects  of  the  nuclein 
upon  some  organ  whose  function  it  is  to  protect  the  body  against 
bacterial  invasion. 

4.  The  longer  the  nuclein  injections  are  continued  and  the  more 
frequently  they  are  administered,  the  more  complete  is  the  immunity 
which  is  secured. 

5.  In  order  to  obtain  this  immunity  the  inoculation  with  the  germ 
must  follow  soon  after  the  last  treatment  with  the  nuclein. 

^Huiti^me  Cangi-l«  International  (F  Hygiene,  2,  44. 


OLANDERS.  101 

Malignant  (Edema. — Kerry  found  that  the  bacillus  of  this  dis- 
ease decomposes  albumin  with  the  formation  of  fatty  acids,  leucin, 
hydroparacumaric  acid,  and  a  foul-smelling  oil  of  the  composition 
CgHjgO^.  This  oil  is  insoluble  in  water,  alkalis,  and  acids,  easily 
soluble  in  ether,  benzol,  bisulphid  of  carbon,  and  alcohol.  It  is 
optically  inactive  and  on  being  oxidized  forms  valerianic  acid. 
Nothing  is  said  concerning  its  action  upon  animals.  Among  the 
gaseous  products  are  carbonic  acid,  hydrogen,  and  marsh  gas.  The 
author  was  unable  to  determine  whether  or  not  free  nitrogen  was 
present. 

Puerperal  Fever. — Bourget  concludes  a  research  on  this  disease 
with  the  following  statements  : 

1.  In  puerperal  fever  the  urine  contains  highly  poisonous  bases. 

2.  The  toxicity  of  the  urine  is  most  marked  when  the  symptoms 
of  the  disease  are  most  grave,  and  diminishes  as  the  symptoms  abate. 

3.  The  ptomains  obtained  from  the  urine  prove  fatal  when  injected 
into  frogs  and  guinea-pigs. 

4.  Toxic  bases,  resembling  those  obtained  from  the  urine,  were 
extracted  from  the  viscera  of  a  woman  who  had  died  of  puerperal 
fever. 

Glanders. — The  toxin  of  this  disease  is  contained  in  the  bacterial 
cell,  and  is  known  as  mallein  or  morvin.  Sterilized  cultures  of  the 
glanders  bacillus  containing  the  chemical  poison  are  used  in  horses 
in  the  same  way  that  tuberculin  is  employed  to  diagnose  tuberculosis 
in  cows.  In  glandered  horses  the  subcutaneous  injection  of  small 
quantities  of  mallein  causes  a  more  marked  elevation  of  temperature 
than  it  does  in  healthy  animals.  However,  Schattenfroh  has  shown 
that  a  like  effect  is  produced  in  glandered  horses  by  the  injection  of 
toxins  from  other  bacteria.  Commercial  mallein  is  prepared  in  a 
number  of  ways,  one  of  which  is  as  follows  :  Growths  of  the  glan- 
ders bacillus,  from  ten  to  fourteen  days  old,  on  potatoes,  are  removed 
with  a  sterilized  spatula  and  rubbed  up  with  sterilized  water  in  the 
proportion  of  one  part  of  the  moist  bacillus  to  nine  parts  of  water. 
This  emulsion  is  allowed  to  stand  for  twenty-four  hours,  and  then 
heated  to  110°  for  fifteen  minutes;  next,  it  is  filtered  through  por- 
celain, 30  per  cent,  of  glycerin  added,  and  concentrated  at  low  tem- 
perature on  the  water-bath  to  one-eighth  of  the  original  volume. 
This  is  again  sterilized  at  110°  and  the  preparation,  now  ready  for 
use,  consists  of  a  clear,  yellowish,  odorless  fluid  of  feebly  acid  or 
neutral  reaction.  Kressling  found  in  mallein  prepared  by  the  above 
method  peptons,  globulins,  xanthin,  guanin,  small  quantities  of  tyro- 
sin  and  leucin,  and  traces  of  volatile  fatty  acids  and  ammonia. 


CHAPTER  V. 

THE  GERMICIDAL  PROPERTIES  OF  BLOOD  SERUM. 

As  early  as  1872  Lewis  and  Cunningham  demonstrated  the  fact 
that  bacteria  injected  into  the  circulation  rapidly  disappear.  In  the 
blood  of  twelve  animals  that  had  been  treated  with  such  injections 
bacteria  could  be  found  after  six  hours  in  only  seven.  In  a  second 
series  of  thirty,  bacteria  were  found  after  twenty-four  hours  in  the 
blood  of  only  fourteen,  and  in  a  third  experiment  involving  seven- 
teen animals,  bacteria  were  found  in  only  two,  when  the  examination 
was  made  from  two  to  seven  days  after  the  injection. 

In  1874  Traube  and  Gscheidlen  found  that  arterial  blood,  taken 
under  aseptic  precautions,  from  rabbits  into  the  jugular  vein  of 
which  one  and  one-half  c.c.  of  a  fluid  rich  in  putrefactive  germs  had 
been  injected  forty-eight  hours  previously,  failed  to  undergo  decom- 
position for  months.  These  investigators  attributed  the  germicidal 
properties  of  the  blood  to  the  ozonized  oxygen.  Similar  results 
were  obtained  by  Fodor  and  by  Wyssokowitsch.  The  latter  accounted 
for  the  disappearance  of  the  germs,  not  by  supposing  that  they  were 
destroyed  in  the  blood,  but  that  they  found  lodgment  in  the  capil- 
laries. 

The  first  experiments  made  with  extra-vascular  blood  were  con- 
ducted by  Grohmann  under  the  direction  of  A.  Schmidt  in  his  re- 
searches upon  the  cause  of  coagulation.  It  was  found  that  anthrax 
bacilli,  after  being  kept  in  plasma,  were  less  virulent,  as  was  demon- 
strated by  their  effect  upon  rabbits,  and  Grohmann  supposed  that  in 
some  way  the  bacteria  were  influenced  by  the  process  of  coagulation. 

In  1887  Fodor  made  a  second  contribution  to  this  subject,  and  in 
this  he  combated  the  retention  theory  of  Wyssokowitsch.  One 
minute  after  the  injection  of  one  c.c.  of  anthrax  culture  into  the  jugu- 
lar vein,  in  eight  samples  of  blood,  Fodor  found  only  one  colony  of 
the  bacillus.  He  then  took  the  blood  from  the  heart  with  a  sterilized 
pipette  and  added  anthrax  bacilli  to  it.  This  was  kept  at  38°,  and 
plates  made  from  time  to  time  showed  a  rapid  diminution  of  the 
number  of  germs  ;  after  a  time,  when  the  blood  had  lost  its  germi- 
cidal properties,  the  number  of  bacteria  began  to  increase. 

In  1888,  Nuttall,  working  under  the  direction  of  Fliigge,  used 
defibrinated  blood  taken  from  various  species  of  animals — rabbits, 
mice,  pigeons,  and  sheep — and  found  that  this  blood  destroyed  the 
bacillus  anthracis,  bacillus  subtilis,  bacillus  megaterium  and  staphyl- 
ococcus pyogenes  aureus  when  brought  in  contact  with  them.     He 

102 


BLOOD  SERUM.  103 

also  confirmed  the  finding  of  Fodor  that  after  a  while  the  blood 
loses  its  germicidal  properties  and  becomes  a  suitable  culture 
medium  in  which  germs  grow  abundantly. 

Nissen  continued  this  work  under  Fliigge's  direction  and  reached 
the  following  conclusions  : 

1.  The  addition  of  small  quantities  of  sterilized  salt  solution  or 
bouillon  to  the  blood  does  not  destroy  its  germicidal  properties. 

2.  Cholera  germs  and  Eberth  bacilli  are  easily  destroyed  by  fresh 
blood. 

3.  In  a  given  volume  of  blood  there  is  a  maximum  number  of 
bacilli  which  can  be  destroyed. 

4.  Blood,  the  coagulability  of  which  has  been  destroyed  by  the 
injection  of  pepton,  is  still  germicidal. 

5.  Blood  in  which  coagulation  is  prevented  by  the  addition  of 
25  per  cent,  of  magnesium  sulphate  has  its  germicidal  properties 
decreased. 

6.  Filtered  blood  plasma  from  the  horse  is  germicidal. 

At  one  time,  Behring  attributed  the  action  of  the  blood  of  the 
white  rat  on  anthrax  bacilli  to  its  great  alkalinity.  He  made  a 
number  of  titrations,  by  which  he  showed  that  the  blood  serum  of 
the  rat  is  somewhat  more  alkaline  than  that  of  certain  animals  which 
are  more  susceptible  to  anthrax,  such  as  the  rabbit,  the  guinea-pig 
and  the  cow.  This  deduction  is  not  justified,  because  there  are 
many  other  and  more  important  points  in  which  these  animals  differ 
from  the  white  rat  than  in  slight  differences  in  the  alkalinity  of  the 
blood  serum.  Had  he  shown  that  the  blood  of  the  adult  rat,  which 
is  not  susceptible  to  anthrax,  is  more  alkaline  than  that  of  the 
young  rat,  which  is  susceptible,  his  argument  would  have  been  more 
plausible  ;  but  even  then  it  would  not  have  deserved  the  dignity  of 
positive  evidence. 

In  1890  Buchner  and  his  students  made  a  valuable  contribution 
to  our  knowledge  of  the  germicidal  properties  of  blood,  and  reached 
the  following  conclusions : 

1.  The  germicidal  action  of  the  blood  is  not  due  to  phagocytes, 
because  it  is  not  influenced  by  the  alternate  freezing  and  thawing  of 
the  blood,  by  which  the  leucocytes  are  destroyed. 

2.  The  germicidal  properties  of  the  cell-free  serum  must  be  due  to 
its  soluble  constituents. 

3.  Neither  neutralization  of  the  serum,  nor  the  addition  of  pepsin, 
nor  the  removal  of  carbon  dioxide  gas,  nor  treatment  with  oxygen, 
has  any  effect  upon  the  germicidal  properties  of  the  blood. 

4.  Dialysis  of  the  serum  against  water  destroys  its  activity,  while 
dialysis  against  0.75  per  cent,  salt  solution  does  not.  In  the  diffu- 
sate  there  is  no  germicidal  substance.  The  loss  by  dialysis  with  water 
must  be  due  to  the  withdrawal  of  the  inorganic  salts  of  the  serum. 

5.  The  same  is  shown  to  be  the  case  when  the  serum  is  diluted 


104  GERMICIDAL  PROPERTIES  OF  BLOOD  SERUM. 

with  water  and  when  it  is  diluted  with  salt  solution ;  in  the  former 
instance  the  germicidal  action  being  destroyed,  while  in  the  latter  it 
is  not. 

6.  The  inorganic  salts  have  in  and  of  themselves  no  germicidal 
action.  They  are  active  only  in  so  far  as  they  affect  the  normal 
properties  of  the  albuminates  of  the  serum.  The  germicidal  prop- 
erties of  the  serum  reside  in  its  albumin  constituents. 

7.  The  difference  in  the  effects  of  active  serum  and  that  which  has 
been  heated  to  55°  is  due  to  the  altered  condition  of  the  albuminate. 
The  difference  may  possibly  be  a  chemical  one  (due  to  changes 
within  the  molecule)  or  it  may  be  due  to  alterations  in  mycelial 
structure.  The  albuminous  bodies  act  upon  the  bacteria  only  when 
the  former  are  in  an  active  state. 

In  the  third  edition  of  this  book  we  pointed  out  an  inconsistency 
between  Buchner's  experimental  results  and  his  conclusions.  Ex- 
perimentally he  ascertained  that  peptic  digestion  of  blood  serum  does 
not  destroy  its  germicidal  properties,  and  yet  he  concluded  that  the 
active  principle  is  serum  albumin.  Since  serum  albumin  is  destroyed 
by  peptic  digestion,  it  cannot  be  the  active  germicidal  agent  in  the 
serum. 

Prudden  found  that  ascitic  and  hydrocele  fluids  restrain  the  de- 
velopment of  certain  germs.  Rovighi  reported  that  the  germicidal 
action  of  the  blood  is  increased  in  febrile  conditions.  Pekelharing 
enclosed  anthrax  spores  in  bits  of  parchment  and  introduced  them 
under  the  skin  of  rabbits.  Thus  treated,  the  spores  soon  lost  their 
virulence  and  finally  their  capability  of  growth.  The  destruction  of 
these  spores  could  not  have  been  due  to  phagocytes,  which  did  not 
penetrate  the  parchment,  but  must  have  been  caused  by  soluble  germ- 
icides. Behring  and  Nissen  found  that  the  serum  of  the  white  rat 
and  of  the  rabbit  destroys  anthrax  bacilli,  while  serum  obtained  from 
the  mouse,  sheep,  guinea-pig,  chicken,  pigeon,  and  frog,  has  no  such 
effect.  It  will  be  observed  from  this  that  there  is  no  constant  rela- 
tion between  the  germicidal  action  of  the  blood  of  animals  of  differ- 
ent species  and  their  susceptibility  to  the  disease  caused  by  the  germ ; 
thus,  the  rabbit  is  highly  susceptible  to  anthrax,  notwithstanding  the 
fact  that  its  blood  destroys  large  numbers  of  these  germs ;  while  the 
chicken  is  immune  to  anthrax  from  the  moment  when  it  comes  from 
the  shell  and  yet  the  bacillus  anthracis  grows  luxuriantly  in  the 
extra- vascular  blood  of  the  chick.  This  demonstrates  that  there  is  a 
great  difference  between  the  action  of  extra-vascular  blood  and  that 
existing  in  the  body,  constantly  fed,  and,  in  case  of  need,  altered  in 
composition  by  certain  glands. 

Halliburton  prepared  from  the  lymphatic  glands  a  globulin  which 
he  designated  cell-globulin-/?,  and  which  agrees  with  fibrin  ferment 
in  inducing  coagulation  in  plasma,  Hankin  tested  the  germicidal 
properties  of  this  globulin,  conducting  his  experiments  in  the  follow- 


BLOOD  SERUM.  105 

ing  manner  :  The  lymphatic  glands  or  the  spleen  of  a  dog,  or  cat,  are 
freed  as  much  as  possible  from  fat  and  connective  tissue,  then  divided 
and  extracted  with  a  dilute  solution  of  sodium  sulphate  (one  part  of 
a  saturated  solution  to  nine  parts  of  water).  The  cell  globulin  passes 
into  solution,  while  the  other  proteids  are  but  sparingly  soluble. 
After  twenty-four  hours,  the  fluid  is  filtered  and  mixed  with  an  ex- 
cess of  alcohol.  The  voluminous  precipitate  contains  the  cell  glob- 
ulin and  is  collected  on  a  filter  and  washed  with  absolute  alcohol. 
For  use,  a  part  is  dissolved  in  water,  and  a  small  quantity  of  a  bouil- 
lon culture  of  the  anthrax  bacillus  is  added.  From  time  to  time 
plate  cultures  are  made,  along  with  control  plates,  and  in  this  way 
the  germicidal  properties  of  the  substance  are  demonstrated.  Hankin 
reached  the  following  conclusions  : 

1 .  Halliburton's  cell-globulin-/9  has  marked  germicidal  properties. 

2.  In  this  respect  it  differs  from  fibrin  ferment. 

3.  The  germicidal  properties  of  this  substance  seem  to  be  identi- 
cal with  those  of  serum,  as  described  by  Buchner,  Nissen  and  Nut- 
tall. 

4.  The  active  properties  of  the  serum  are  probably  due  to  this  or 
an  allied  body. 

Bitter  repeated  these  experiments,  but  failed  to  confirm  them ; 
however,  it  is  certain  that  the  spleen  contains  a  germicidal  substance, 
but  whether  it  can  be  extracted  by  the  method  of  Hankin  or  not  we 
do  not  know. 

Christmas  prepared  a  germicidal  substance  from  the  spleen  and 
other  organs  by  the  following  method :  The  animal  is  killed  with 
ether,  opened  under  aseptic  precautions,  and  the  organ  removed,  cut 
into  fine  pieces,  covered  with  50  c.c.  of  glycerin  and  allowed  to  stand 
for  twenty-four  hours  and  then  filtered.  The  filtrate  is  precipitated 
with  five  times  its  volume  of  alcohol  and  this  is  immediately  decanted. 
The  precipitate  is  washed  with  absolute  alcohol  in  order  to  remove 
the  glycerin,  and  then  traces  of  alcohol  are  taken  up  by  pressure 
between  folds  of  blotting  paper  and  the  precipitate  is  dissolved  in 
25  c.c.  of  distilled  water.  Through  this  solution  air  is  passed  for 
some  hours  in  order  to  remove  traces  of  alcohol,  and  then  the  fluid 
is  filtered  and  its  germicidal  action  tested. 

Bitter  examined  this  method  also,  and  the  impartial  reader  may 
see  that  he  did  not  do  so  with  fairness.  However,  this  fact  renders 
the  work  all  the  more  valuable  because  his  results  confirm  the  state- 
ments of  Christmas.  Bitter  killed  his  animals  by  venesection  and, 
in  some  cases,  at  least,  prepared  the  substance  in  unsterilized  vessels, 
but  even  when  this  was  done  the  solution  was  germ-free  and  mani- 
fested marked  germicidal  properties.  However,  Bitter  found  a  dif- 
ference between  this  substance  and  the  germicidal  constituent  of 
blood  serum ;  the  latter  is  certainly  destroyed  by  a  temperature  of 
65°,  while  the  solution  of  Christmas,  after  having  been  heated  to  this 


106  GERMICIDAL  PROPERTIES  OF  BLOOD  SERUM. 

temperature,  is  still  capable  of  destroying  from  35,000  to  40,000 
typhoid  bacilli  within  four  hours. 

It  is  possible  that  the  more  powerful  action  of  the  solution  made 
by  Christmas  is  due  to  the  presence  of  the  germicidal  substance  in 
more  nearly  a  chemically  pure  condition  than  it  exists  in  blood 
serum,  and  later  we  shall  see  that  the  temperature  at  which  the 
germicidal  activity  of  blood  serum  is  arrested  is  variable,  and  de- 
pends upon  conditions  which  are  not  thoroughly  understood. 

Attempts  have  been  made  to  determine  the  nature  of  the  germi- 
cidal constituent  by  the  action  of  precipitating  reagents  on  the  pro- 
teids  of  blood  serum.  Buchner  was  not  able  to  obtain  a  germicidal 
solution  by  precipitating  all  the  proteids  with  absolute  alcohol,  free- 
ing the  precipitate  from  alcohol,  drying  it,  and  then  redissolving  it. 
Inasmuch  as  he  failed  to  give  the  methods  employed  in  freeing  the 
precipitate  from  alcohol,  the  temperature  or  the  conditions  under 
which  it  was  dried,  and  the  nature  of  the  menstruum  by  which  re-so- 
lution was  effected,  his  conclusion  that  alcohol  destroys  the  germi- 
cidal substance  must  remain  open  to  question.  On  the  other  hand, 
Christmas  found  that  when  the  proteids  are  precipitated  with  alcohol 
and  the  precipitate  dissolved  in  a  volume  of  water  equal  to  that  of 
the  original  solution,  the  solution  thus  obtained  has  a  more  powerful 
germicidal  action  than  the  serum. 

Bitter  reached  the  conclusion  that  anthrax  and  typhoid  bacilli  are 
destroyed  by  "  precipitated  serum,"  but  not  so  energetically  as  by 
normal  serum.  Emmerich,  Tsuboi,  Steinmetz  and  Low  studied  the 
effect  of  precipitation  of  the  proteids  upon  the  germicidal  properties 
of  the  blood  serum.  An  active  serum  was  dialyzed  in  a  sterilized 
parchment  paper  tube  against  water  for  from  twelve  to  eighteen 
hours.  By  the  expiration  of  that  time  the  serum  globulin,  becoming 
insoluble  on  account  of  the  withdrawal  of  inorganic  salts,  was  depos- 
ited. The  dialyzer  was  dried  with  sterilized  filter  paper,  and  the 
globulin-free  serum  was  precipitated  with  several  volumes  of  alcohol. 
The  precipitate  was  collected  on  a  sterilized  filter  and  the  alcohol 
removed  by  sterilized  porous  plates  and  filter  paper.  The  precipi- 
tate was  then  finely  divided,  dried  for  half  an  hour  in  vacuo  at  36°, 
then  rubbed  up  in  a  sterilized  mortar  and  dissolved  in  sterilized 
water,  to  which  salt  solution  had  been  added.  In  the  solution  thus 
prepared  germs  did  not  show,  after  from  three  to  four  hours,  either 
a  marked  increase  or  decrease,  but  when  the  solution  was  heated  to 
100°,  allowed  to  cool,  and  then  inoculated  with  germs,  the  increase 
was  four  hundred-fold  within  four  hours.  It  was  next  found  that  if 
instead  of  water  a  0.05  per  cent,  aqueous  solution  of  potassic  hydrate 
was  employed  in  dissolving  the  alcoholic  precipitate  in  the  globulin- 
free  serum,  this  solution  possessed  all  the  germicidal  strength  of  the 
original  serum.  The  same  was  found  to  be  true  of  dilute  alkaline 
solutions  of  the  alcohol  precipitate  in  serum  from  which  the  globulin 


BLOOD  SERUM.  107 

had  not  been  removed.  The  dilute  alkaline  solution  was  shown  not 
to  have  any  germicidal  action  in  and  of  itself.  From  these  experi- 
ments the  above-mentioned  investigators  concluded  that  the  germi- 
cidal constituent  of  blood  serum  is  an  alkaline  compound  of  serum 
albumin.  They  also  found  that  heating  the  serum  albumin  alkaline 
solution  to  65°,  or  higher  destroyed  its  germicidal  action,  and  they 
explained  this  effect  of  heat  upon  blood  serum  and  other  artificial 
solutions  by  supposing  that  the  high  temperature  breaks  up  the  com- 
bination of  the  alkali  with  the  serum  albumin.  Furthermore,  they 
found  that  a  serum  that  had  been  rendered  inactive  by  a  temperature 
of  55°  could  be  regenerated,  in  part  at  least,  by  the  addition  of  a 
small  amount  of  alkaline  menstruum. 

Since  Fodor  and  Ziintz  have  shown  that  freshly  drawn  blood 
rapidly  decreases  in  alkalinity  on  standing  in  vitro,  an  explanation  of 
the  fact  that  blood  serum  rapidly  loses  its  germicidal  properties 
naturally  suggests  itself.  Emmerich  and  his  co-workers  confirmed 
their  belief  in  this  theory  by  demonstrating  that  blood  serum  which 
has  been  rendered  feebly  acid  (0.67  part  of  sulphuric  acid  per  mille) 
has  no  germicidal  action,  but  furnishes  a  good  culture  medium. 
These  investigators  show  the  important  role  that  the  small  amount 
of  alkali  plays  in  the  germicidal  action  of  blood  serum.  This  had 
already  been  demonstrated  by  Fodor  by  quite  a  different  line  of 
investigation.  The  latter  found  that  the  resistance  of  rabbits  to 
anthrax  is  markedly  increased  by  the  administration,  by  stomach 
or  subcutaneously,  of  sodium  phosphate,  carbonate  or  bicarbonate, 
or  of  potassium  carbonate.  Low  concludes  that  the  introduction  of 
alkali  into  the  albumin  molecule  increases  its  lability,  and  he  cites 
examples  from  organic  chemistry  in  support  of  this  view.  Em- 
merich and  his  assistants  think  it  highly  probable  that  only  a  com- 
paratively small  part  of  the  albumin  is  active,  and  this  small  part, 
they  suppose,  originates  in  the  albumin  of  the  daily  food  which  is 
converted  into  lymph  cells,  and  by  the  disintegration  of  these  it 
passes  into  solution  in  the  blood ;  however,  they  admit  that  there 
are  some  reasons  for  believing,  with  Buchner,  that  the  whole  of  the 
serum  albumin  is  active.  They  state  that  it  is  possible,  but  highly 
improbable,  that  the  germicidal  substance  is  not  the  serum  albumin, 
but  some  substance  that  is  precipitated  along  with  this  by  alcohol 
and  other  agents. 

In  1893  Vaughan  and  McClintock,  after  reviewing  the  literature, 
reported  their  work  on  the  germicidal  constituent  of  blood  serum  as 
follows : 

1.  The  serum  albumin  is  not  the  germicidal  substance  in  blood 
serum.  Either  this  must  be  true  or  the  experiment  by  which  Buch- 
ner demonstrated  that  an  active  pepsin  does  not  destroy  the  germici- 
dal action  of  blood  serum  must  have  been  an  error ;  because  peptic 
digestion  readily  and  completely  converts  serum  albumin  into  pep- 


108  GERMICIDAL  PROPERTIES  OF  BLOOD  SERUM. 

tons,  and  we  know  that  peptons  are  especially  favorable  to  bacterial 
growth. 

2.  The  germicidal  substance  must  belong  to  the  proteids.  Other- 
wise it  would  be  difficult  to  explain  the  fact  that  a  temperature  of 
55"  renders  blood  serum  inactive. 

3.  The  only  proteid  likely  to  be  present  in  blood  serum  and  which 
is  not  destroyed  by  peptic  digestion  is  nuclein.  Having  reached 
these  conclusions,  the  following  questions  naturally  presented  them- 
selves : 

1.  Is  there  a  nuclein  in  blood  serum?  2.  Has  this  nuclein,  if 
there  be  one,  germicidal  properties  ? 

The  first  of  these  questions  was  answered  in  the  following  manner  : 
An  active  blood  serum  was  treated  with  ten  times  its  volume  of  a 
mixture  of  equal  parts  of  absolute  alcohol  and  ether.  This  produced 
a  voluminous  precipitate  which,  after  repeated  washings  with  alcohol 
and  ether,  was  submitted  to  the  action  of  pepsin-hydrochloric  acid 
in  the  incubator  at  38°.  Digestion  was  continued  until  the  super- 
natant fluid  failed  to  respond  to  the  biuret  test  for  peptons.  Each 
time  this  test  was  made  the  fluid  was  decanted  from  the  undigested 
part  and  replaced  by  an  equal  volume  of  fresh  digestive  fluid.  In 
all  cases,  digestion  was  prompt  and  proceeded  to  a  certain  point, 
when  it  ceased  altogether.  The  undigested  proteid  was  small  in 
amount  and  grayish  in  color.  This  was  collected  on  a  small  steril- 
ized filter,  and  washed  first  with  a  0.2  per  cent,  solution  of  hydro- 
chloric acid,  and  then  with  alcohol.  After  the  washing  with  alcohol, 
the  filter  was  allowed  to  stand  exposed  to  the  air  for  half  an  hour  or 
longer  in  order  that  all  of  the  alcohol  might  pass  through  or  evap- 
orate. The  precipitate  was  then  dissolved  in  a  sterilized  solution  of 
potassic  hydrate.  The  strength  of  this  alkaline  solution  usually  em- 
ployed was  0.12  per  cent,  and  the  solution  contained  in  addition  to 
the  alkali  0.6  per  cent,  of  sodium  chlorid.  In  some  instances  a 
solution  containing  1.2  grams  of  potassic  hydrate,  six  grams  of 
sodium  chlorid,  and  one  gram  each  of  sodium  bicarbonate  and  diso- 
dium  hydrogen  phosphate  to  one  liter  of  water,  was  employed  as  a 
solvent.  The  solution  was  filtered  through  a  Chamberland  tube  and 
received  in  a  sterilized  flask.  The  solution  thus  obtained  was  per- 
fectly clear,  colorless,  and  did  not  respond  to  the  biuret  test.  The 
addition  of  strong  nitric  acid  produced  a  cloudiness,  which  dissolved  * 
on  the  further  addition  of  the  acid.  This  acid  solution  did  not  be- 
come yellow  on  being  heated,  but  did  so  after  the  addition  of  am- 
monia. It  is  true  that  there  was  no  ultimate  analysis  made  of  this 
substance,  but  the  above  experiments  demonstrate  the  fact  that  blood 
serum  contains  a  proteid  which  resists  peptic  digestion,  does  not  re- 
spond to  the  biuret  and  xanthoproteic  tests,  and  which,  as  we  shall 
see  later,  has  marked  germicidal  properties.  Is  this  substance  serum 
albumin  or  is  it  a  nuclein  ?     Moreover,  the  presence  of  nucleinic  acid 


BLOOD  SERUM.  109 

in  blood  serum  has  been  confirmed  by  the  work  of  Lilienfeld,  whose 
paper  was  published  in  1895,  while  that  of  the  American  investi- 
gators was  published  in  1893. 

The  germicidal  action  of  the  nuclein,  obtained  as  above  stated, 
from  blood  serum,  was  abundantly  demonstrated  by  a  long  series  of 
experiments  published  in  the  original  contributions  on  this  subject 
and  in  part  reproduced  in  the  third  edition  of  this  book. 

In  the  third  edition  of  this  book  there  occurred  the  following 
paragraph  :  "  The  origin  of  the  nuclein  now  found  for  the  first  time 
in  blood  serum  is  an  interesting  question.  Does  it  come  from  the  dis- 
integration of  the  poly  nuclear  cells,  or  shall  we  regard  certain  white 
blood  corpuscles  as  unicellular  organisms  whose  function  it  is  to 
secrete  this  nuclein  ?  "  It  will  be  seen  from  what  is  to  follow  that 
practically  all  the  investigations,  which  have  been  carried  on  since 
the  above  paragraph  was  written,  have  had  for  their  object  the  solu- 
tion of  the  questions  there  proposed. 

Buchner  has  long  used  the  term  alexins  to  designate  the  germi- 
cidal substance  or  substances  that  exist  in  blood  serum  and  whose 
exact  nature  has  not  yet  been  determined.  His  most  potent  argu- 
ment against  alexin  being  a  nuclein  lies  in  the  fact  that  when  blood 
serum  is  heated  to  56°  or  58°  it  loses  its  bactericidal  properties; 
while  it  is  well  known  that  aqueous  solutions  of  nucleinic  acid  are 
not  altered  by  much  higher  temperatures.  A  serum  whose  germi- 
cidal action  has  been  destroyed  by  heat  is  designated  as  inactive. 
It  is  within  the  range  of  possibility  that  there  may  be  a  nuclein  or  a 
nucleinic  acid  so  labile  that  it  loses  its  germicidal  action  at  the  rela- 
tively low  temperature  mentioned  above.  It  is  also  possible  that 
while  an  aqueous  solution  of  nucleinic  acid  may  retain  its  germicidal 
properties  at  100°,  when  mixed  with  the  constituents  of  blood 
serum  it  may  be  altered  at  a  much  lower  temperature.  It  will  be 
seen  from  this  that  the  effect  of  temperature  upon  the  alexins  of 
the  blood  is  a  matter  of  importance  inasmuch  as  it  may  be  suggestive 
of  the  composition  of  these  bodies.  Bail  ^  in  his  studies  on  the 
action  of  leukocidin  found  that  while  this  toxin  dissolves  or  disinte- 
grates the  leucocytes,  it  does  not  destroy  the  bactericidal  substance 
contained  in  them  and  that  when  this  phenomenon  occurs  the  ger- 
micidal constituent  of  the  white  blood  corpuscle  passes  into  solution, 
and  that  such  a  solution  may  be  boiled  without  losing  its  germicidal 
properties.  He  also  ascertained  that  when  isolated  leucocytes  are  ex- 
tracted with  alkaline  sodium  chlorid  solution  and  this  extract  precipi- 
tated with  acetic  acid  and  then  redissolved  in  an  alkaline  inactive  di- 
lute blood  serum,  a  germicidal  solution  is  obtained  and  that  this  may 
be  heated  for  one-half  hour  to  85°  without  loss  of  bactericidal  prop- 
erties. Furthermore,  he  showed  that  the  germicidal  properties  were 
due  to  the  substance  precipitated  with  acetic  acid  inasmuch  as  he 
^  Archiv  f.  Hygiene,  30  and  32. 


110  GERMICIDAL  PROPERTIES  OF  BLOOD  SERUM. 

found  that  on  removal  of  this  precipitate  the  solution  became  inac- 
tive. Lowit  ^  rubbed  up  isolated  leucocytes  with  pulverized  glass 
and  extracted  from  the  powder  thus  obtained  a  bactericidal  sub- 
stance which  is  not  destroyed  or  even  weakened  in  action  by  five 
minutes'  boiling.  The  germicidal  extract  thus  obtained  is  cloudy,  of 
feebly  alkaline  reaction,  gives  the  biuret  test,  contains  but  little 
proteid  which  is  precipitable  by  heat,  and  yields  a  flocculent  precip- 
itate on  the  addition  of  acetic  acid.  From  their  investigations  both 
Bail  and  Lowit  conclude  that  the  heat-resisting  bactericidal  substance 
obtained  by  them  is  nuclein  or  nucleinic  acid  or  a  derivative  of  one  of 
these  substances.  Schattenfroh  ^  combats  the  conclusion  reached  by 
the  above-mentioned  authors.  He  claims  that  the  germicidal  sub- 
stance obtained  by  Lowit  consists  of  sodium  silicate  extracted  from 
the  powdered  glass,  and  he  attempts  to  demonstrate  this  by  an  ex- 
periment in  which  the  germicidal  action  of  this  compound  is  tested. 
This  experiment,  however,  is  not  convincing,  inasmuch  as  it  shows 
that  even  a  one  per  cent,  solution  of  sodium  silicate  is  practically 
without  bactericidal  properties  ;  and  it  is  not  probable  that  water  or 
physiological  salt  solution  will  extract  from  glass  a  stronger  solu- 
tion than  this.  In  his  criticism  on  the  work  of  Bail,  Schattenfroh 
admits  that  the  results  obtained  by  his  opponent  are  exactly  what 
he  himself  observed,  but  he  thinks  that  in  this  case  the  heat-resist- 
ing germicidal  substance  is  the  alkali  used  in  extracting  the  leuco- 
cytes. This  criticism  also  is  somewhat  far-fetched  inasmuch  as 
Schattenfroh  attempts  to  demonstrate  the  truth  of  his  assertion  by 
showing  that  Bail's  extract,  when  neutralized  or  rendered  feebly 
acid,  is  without  germicidal  properties,  but  everyone  knows  that 
blood  serum  when  neutralized  or  rendered  feebly  acid  also  becomes 
inactive.  Moreover,  Fodor  long  ago  demonstrated  that  increasing 
the  alkalinity  of  the  blood  increases  its  germicidal  properties. 
Schattenfroh's  own  investigations  have  demonstrated  that  a  tempera- 
ture of  56°  to  58°  does  not  destroy  the  alexins  in  aqueous  extracts 
of  isolated  leucocytes.  On  this  point  he  makes  the  following  state- 
ment :  "  Experiment  3  and  others  leave  no  longer  room  for  doubt 
that  the  bactericidal  action  of  leucocytes  is  retained  after  being 
heated  in  distilled  water  for  half  an  hour  to  from  55°  to  60°.  It 
may  be  seen  from  what  has  been  stated  that  the  resistance  of  the 
alexins  to  heat  is  largely  dependent  upon  the  nature  of  the  menstruum 
in  which  they  are  dissolved  or  held  in  suspension." 

The  evidence  concerning  the  manner  in  which  the  leucocytes  pro- 
duce alexins  is  scarcely  more  satisfactory  than  that  bearing  upon  the 
influence  of  temperature  upon  the  latter.  Haukin '  was  probably 
the  first  to  suggest  that  the  alexins  are  products  of  the  amphophil 

^Ziegler's  Beitrdge,  22. 
^Archivf.  Hygiene,  35. 
^  CerUralblatt  f,  Bakterioloqie,  12. 


BLOOD  SEBUM.  Ill 

leucocytes,  which  he  proposes  should  be  called  alexocytes.  Accord- 
ing to  his  theory  the  pseudo-eosinophil  granules  constitute  the  parent 
substance  of  the  alexins.  He  succeeded  in  diminishing  the  number 
of  these  leucocytes  in  the  blood  by  the  intravenous  injection  of  leech 
extract  and  found  that  the  blood  thus  obtained  was  not  equivalent  to 
normal  blood  in  its  germicidal  properties.  Denys  ^  and  his  students 
were  among  the  earlier  advocates  of  the  theory  that  the  alexins  are 
secretion  products  of  the  leucocytes.  Buchner  ^  obtained  leucocytes 
in  relatively  large  numbers  by  introducing,  under  aseptic  precautions, 
wheat  gluten  or  aleuron  into  the  pleural  cavities  of  animals ;  an 
exudate  rich  in  white  blood  corpuscles  forms  about  the  foreign  body 
after  a  few  hours  and  the  corpuscles  thus  obtained  serve  for  experi- 
mentation. The  purpose  of  Buchner's  first  experiments  was  to  com- 
bat the  phagocytic  theory  of  Metschnikoff,  and  he  demonstrated  that 
the  leucocytes,  obtained  as  stated  above,  when  destroyed  by  alternate 
freezing  and  thawing,  markedly  increased  the  germicidal  action  of 
the  serum  in  which  they  were  suspended,  while  at  the  same  time  they 
were  deprived  of  life.  In  other  words,  these  experiments  demon- 
strated that  the  disintegration  of  white  blood  corpuscles  liberates  a 
germicidal  substance.  Leucocytes  obtained  after  Buchner's  method 
can  be  freed  from  serum  in  the  centrifuge,  and  may  be  dissolved  or 
suspended  in  physiological  salt  solution,  distilled  water,  or  in  either 
an  active  or  an  inactive  serum.  By  experiments  of  this  kind  Hahn' 
demonstrated  that  the  addition  of  leucocytes  to  an  active  serum  in- 
creased the  activity  of  the  latter,  while  when  added  to  an  inactive 
serum  they  restored  its  activity.  In  this  way  it  has  been  shown 
beyond  controversy  that  blood  serum  owes  its  germicidal  properties 
to  the  white  corpuscles.  Numerous  investigators  have  fully  demon- 
strated that  the  disintegration  of  the  leucocyte  is  followed  by  the 
liberation  of  alexins,  and  the  most  important  question  still  unsettled 
in  this  connection  is  whether  or  not  the  living  white  corpuscles 
secrete  alexins.  Laschtschenko  *  has  shown  that  either  active  or  in- 
active serum  of  the  horse  extracts  alexins  from  the  leucocytes  of  the 
rabbit.  This  experiment  was  made  in  the  following  way :  The 
leucocytes  were  obtained  in  a  pleural  exudate,  prepared  after  the 
method  of  Buchner.  This  exudate  was  placed  in  a  centrifuge  and 
the  corpuscles  separated  from  the  serum.  The  corpuscles  were  then 
repeatedly  washed  in  the  centrifuge  with  physiological  salt  solution 
and  after  they  had  been  thoroughly  separated  from  all  the  constit- 
uents of  the  serum  they  were  well  mixed  with  serum  obtained  from 
other  animals  and  freed  from  their  own  corpuscles  by  means  of  the 
centrifuge.  Both  active  and  inactive  sera  were  used  in  these  experi- 
ments.    The  result,  which  has  already  been  stated,  is  attributed  to 

'  La  cellule,  10,  et  seq. 

2  Munchener  med.  Wochenschrift,  1894. 

^Arckivf.  Hygiene,  25. 

*Archivf.  Hygiene,  37. 


112  GERMICIDAL  PROPERTIES  OF  BLOOD  SERUM. 

the  biological  stimulation  of  the  foreign  serum  on  the  leucocytes  of 
the  rabbit.  It  must  be  admitted,  however,  that  it  is  possible  that  the 
serum  of  the  horse  destroys  the  leucocytes  of  the  rabbit  and  that  the 
germicidal  substances  which  pass  into  solution  in  the  serum  result 
from  the  disintegration  of  the  corpuscles.  From  microscopical  study 
of  the  leucocytes  thus  subjected  to  the  action  of  the  serum  of  the  horse, 
Trommsdorff  ^  concludes  that  the  leucocytes  still  retain  their  vitality 
while  the  alexins  pass  from  them  into  the  serum.  He  finds  that  cor- 
puscles treated  in  this  way  still  possess  amoeboid  movement  and  when 
compared  with  leucocytes  known  to  be  living  show  no  signs  of  dis- 
integration. It  is  therefore  highly  probable  that  the  living  leucocyte 
secretes  a  germicidal  substance. 

We  will  close  our  remarks  upon  the  germicidal  constituent  of  the 
blood  with  the  following  statements  :  (1)  The  exact  nature  of  the 
germicidal  constituents  of  the  blood,  or  alexins,  is  not  known.  (2) 
The  alexins  have  their  origin  in  the  white  blood  corpuscles.  (3)  Dis- 
integration of  the  white  blood  corpuscle  liberates  alexins.  (4)  It  is 
probably  true  that  alexins  are  also  secreted  by  living  leucocytes. 

^Archiv  f.  Hygiene,  40. 


CHAPTER   VI. 

THE  SPECIFIC   PRECIPITINS. 

The  announcement  by  Widal/  that  typhoid  bacilli,  killed  by  a 
heat  of  56°,  are  still  agglutinated  with  the  homologous  serum,  led 
Kraus  ^  to  try  the  effect  of  an  homologous  serum  on  a  filtered  bac- 
terial culture,  and  this  experiment  was  the  beginning  of  our  knowl- 
edge of  the  specific  precipitins.  Cultures  of  the  cholera  bacillus 
were  freed  from  germs  by  filtration  through  porcelain,  and  after 
having  been  proved  to  be  sterile  were  treated  with  different  quanti- 
ties of  a  sterile  cholera  serum.  When  these  two  fluids  were  mixed 
the  mixture  became  cloudy  and  after  a  while  filled  with  fine  floccules 
which  gradually  subsided,  leaving  a  supernatant  clear  fluid.  Fur- 
ther investigation  showed  that  the  germ-free  bacterial  culture  and 
the  serum  must  be  homologous  in  order  to  obtain  this  precipitation. 
A  serum  obtained  from  an  animal  which  has  been  rendered  immune 
to  the  cholera  bacillus  gives  a  precipitate  when  added  to  filtered  cul- 
tures of  the  cholera  bacillus,  but  gives  no  cloudiness  when  added  to 
like  cultures  of  any  other  germ.  It  was  also  found  that  the  serum 
of  non-immunized  animals  does  not  give  precipitates  with  the  filtered 
cultures  of  any  of  the  pathogenic  bacteria.  Kraus  extended  his  in- 
vestigations using  the  sera  and  filtered  cultures  not  only  of  cholera, 
but  also  of  typhoid  fever  and  the  plague,  and  found  that  the  reaction 
observed  by  him  was  a  specific  one  inasmuch  as  it  was  obtained  only 
with  homologous  sera  and  bacterial  filtrates,  and  not  with  heterolo- 
gous fluids.  Furthermore,  he  demonstrated  that  the  substance  in  the 
filtered  culture  to  which  this  reaction  is  due  exists  within  the  bac- 
terial cell.  This  he  did  by  rubbing  up  cholera  bacilli  taken  from 
agar  cultures  with  powdered  glass  and  submitting  this  mixture  to  a 
pressure  of  300  atmospheres ;  the  compressed  mass  was  then  ex- 
tracted with  alkaline  bouillon,  diluted  and  filtered  through  porcelain, 
when  it  was  found  that  this  filtrate  gave  a  precipitate  with  cholera 
serum.  This  shows  that  the  specific  reaction  observed  in  these  experi- 
ments for  the  first  time,  is  due  to  the  presence  in  the  filtrate  of  a  sub- 
stance extracted  from  the  bacterial  cells.  Cultures  filtered  in  the  ordi- 
nary way  through  stone  give  this  reaction  because  filtrates  thus  obtained 
contain  substances  which  originated  within  the  bacterial  cells. 

NicoUe '  repeated,  confirmed  and  extended  the  observations  of 

'  La  Semaine  Med.,  1897. 
*  Wiener  klin.  Wochenschrifi,  1897. 
^  Amxales  deV  Instilut  Pasteur,  12. 
8  113 


114  THE  SPECIFIC  PBEGIPITINS. 

Kraus,  and  subsequent  investigations  by  others  have  shown  that  this 
specific  reaction  holds  good  with  all  bacteria  to  which  animals  have 
been  immunized. 

The  substance  in  the  serum  which  enters  into  this  reaction  is 
known  as  the  precipitin  and  the  precipitate  thus  formed  is  designated 
as  a  precipitum. 

Bordet  found  that  when  milk  which  had  been  partially  sterilized 
by  being  heated  for  one  hour  at  65°  was  injected  intraperitoneally 
and  this  injection  repeated  at  short  intervals  for  some  time,  the 
blood  serum  of  the  animals  thus  treated  gives  with  the  milk  which 
has  been  injected  a  precipitate.  He  placed  three  c.c.  of  the  serum 
from  the  animal  treated  with  the  milk  in  one  test-tube  and  the  sera 
of  other  animals  in  corresponding  tubes  as  controls,  and  found  that 
on  the  addition  of  the  milk  which  had  been  used  in  the  treatment 
of  the  animal,  a  precipitate  appears,  while  no  cloudiness  occurs 
on  the  addition  of  the  same  milk  to  other  sera.  On  the  ad- 
dition of  ten  or  twelve  drops  of  milk  to  the  homologous  serum, 
floccules  form  and  slowly  subside,  leaving  a  clear  supernatant 
fluid,  while  in  the  other  sera  the  milk  diffuses,  forming  opalescent 
homogeneous  fluids.  Further  investigations,  notably  those  of  Was- 
sermann  and  Schiitze,^  have  shown  that  the  serum  of  a  rabbit  treated 
with  cow's  milk  gives  a  precipitate  with  cow's  milk  and  not  with 
the  milk  of  any  other  animal,  and  that  rabbits  treated  with  goat's 
milk  furnish  a  serum  that  gives  precipitates  with  goat's  milk  and 
with  no  other  milk  ;  and  the  same  holds  good  when  the  experiments 
are  made  with  woman's  milk.  By  this  reaction  the  source  of  a  given 
sample  of  milk  can  be  told  with  certainty.  The  following  are  the 
details  of  an  experiment  of  this  kind  :  Several  rabbits  were  treated 
subcutaneously  or  intraperitoneally  at  intervals  of  from  three  to 
four  days  with  from  10  to  50  c.c.  of  cow's  milk,  sterilized  with 
chloroform,  while  a  like  number  were  treated  in  the  same  way  with 
goat's  milk,  and  a  third  lot  treated  with  woman's  milk.  After  the 
rabbits  had  undergone  this  treatment  for  about  three  weeks  and 
each  had  received  about  100  c.c.  of  milk,  they  were  bled  and  their 
sera,  diluted  1:5,  were  added  to  milk,  diluted  1:40,  and  these  mix- 
tures allowed  to  stand  at  room  temperature  for  some  hours.  It  was 
found  that  the  serum  from  the  rabbits  treated  with  cow's  milk  pre- 
cipitated the  albuminous  substances  in  cow's  milk,  but  had  no  such 
action  on  the  milk  of  either  woman  or  goat ;  while  the  serum  of  the 
rabbit  treated  with  goat's  milk  precipitated  the  casein  of  goat's  milk 
and  had  no  effect  upon  that  of  the  woman  or  the  cow,  etc.  The 
serum  of  animals  that  have  been  treated  as  above  described  with 
milk  is  designated  lactoserum,  and  it  has  been  found  that  the 
lactosera  are  in  all  cases  specific  in  their  reactions.  Indeed,  the  dis- 
covery of  this  reaction  has  shown  that  the  proteids  of  the  milk  of 
'  Deutsche  med.  Wochenschrift,  1900. 


PRECIPITINS.  115 

each  species  of  animal  differ  from  those  of  every  other  species,  and 
it  certainly  should  convince  us  that  it  is  impossible  by  any  chemical 
process  to  make  cow's  milk  a  perfect  substitute  for  the  mother's 
milk  in  the  feeding  of  infants.  Fisch  ^  found  that  rabbits  treated 
with  an  emulsion  of  the  cells  of  the  udders  of  animals  furnish  spe- 
cific lactosera,  and  this  demonstrates  that  milk  is  not  a  filtration 
product,  but  that  some  of  its  important  constituents  result  from  the 
transformation  of  the  specific  cells  of  the  gland.  Milk  which  has 
been  boiled  for  half  an  hour  loses  its  power  of  forming  a  precipitum 
on  the  addition  of  its  specific  lactoserum. 

Myers  ^  prepared  crystallized  egg  albumin  from  fresh  eggs  and  by 
repeated  intraperitoneal  injections  of  this  substance  into  rabbits,  he 
obtained  a  serum  that  gives  a  dense  precipitate  when  added  to  so- 
lutions of  crystallized  egg  albumin.  This  precipitate  forms  at  or- 
dinary temperature,  but  its  formation  is  accelerated  at  37°.  It  is 
soluble  in  two  per  cent,  sodium  chlorid  solution  and  such  a  solution 
gives  the  ordinary  proteid  reactions.  The  serum  of  a  rabbit  treated 
with  egg  albumin  from  the  fowl  forms  a  slight  precipitate  with  the 
albumin  obtained  from  ducks'  eggs.  Ovasera  have  no  precipitating 
action  on  globulin  obtained  from  sheep  serum  nor  on  that  from  bul- 
lock serum,  nor  on  serum  albumin  from  the  sheep  or  the  bullock,  nor 
on  pepton.  Myers  next  prepared  serum  globulin  from  the  blood  of 
the  sheep  and  obtained  from  rabbits  treated  with  this  preparation  a 
serum  which  has  no  action  on  egg  albumin  or  upon  pepton,  but  does 
give  a  slight  precipitate  with  globulin  obtained  from  bullock's  serum. 
The  serum  of  a  rabbit  immunized  against  the  globulin  of  the  sheep's 
blood  also  agglutinates  the  red  corpuscle  of  the  sheep.  This  un- 
doubtedly is  due  to  the  presence  in  the  red  blood  corpuscles  of  the 
sheep  of  a  substance  allied  to,  if  not  identical  with,  the  globulin  ob- 
tained from  sheep's  blood.  It  was  also  found  that  the  globulin 
serum  agglutinates  the  washed  red  blood  corpuscles  of  the  fowl. 
From  these  observations  Myers  draws  the  following  conclusions  :  "  It 
follows  from  these  facts  that  the  product  here  called  '  sheep's  glob- 
ulin '  is  a  mixture  of  substances.  The  main  portion  of  the  pre- 
cipitum given  by  its  precipitin  is  formed  from  a  substance  which  is 
not  present  in  the  '  serum  globulin '  of  the  bullock.  One  substance, 
present  in  small  quantities  in  the  sheep's  globulin,  is,  however,  also 
present  in  bullock's  globulin,  since  the  precipitin  of  sheep's  globulin 
gives  a  small  precipitum  with  bullock's  globulin.  This  substance 
common  to  the  two  globulins  is  not  present  in  the  red  corpuscles  of 
the  sheep  or  fowl,  since  the  precipitin  in  bullock's  globulin  does  not 
agglutinate  these  corpuscles.  Further,  we  must  suppose  that  there 
are  two  other  substances  in  the  serum  globulin  of  the  sheep,  one 
present  in  fowl's  red  corpuscles  and  the  other  present  in  those  of  the 

1  St.  Louis  Courier  of  Medicine,  1900. 

2  The  Lancet,  Vol.  II.,  1900. 


116  THE  SPECIFIC  PRECIPITINS. 

sheep,  since,  as  the  following  experiment  shows,  immunization  against 
sheep's  globulin  leads  to  the  appearance  in  the  serum  of  two  distinct 
agglutinins,  for  sheep's  and  for  fowl's  corpuscles,  respectively.  If 
the  serum  of  a  rabbit  immunized  against  sheep's  globulin  be  allowed 
to  agglutinate  washed  corpuscles  of  the  sheep,  the  clear  fluid  left 
above  the  corpuscles  will  not  now  agglutinate  fresh  corpuscles  of  the 
sheep  if  sufficient  corpuscles  have  been  used  in  the  first  instance. 
But  this  fluid  still  has  the  power  of  agglutinating  fowl's  corpuscles. 
And,  conversely,  after  antiglobulin  has  agglutinated  fowl's  cor- 
puscles, it  has  lost  the  power  of  agglutinating  fresh  fowl's  cor- 
puscles, but  will  still  agglutinate  those  of  the  sheep."  Animals 
immunized  with  pepton  furnished  a  serum  which  gave  a  precipitum 
with  pepton  and  failed  to  react  with  any  other  proteid.  The  pepton 
precipitum,  after  being  carefully  washed  with  physiological  salt  so- 
lution and  then  dissolved  in  two  per  cent,  saline  solution,  does  not 
give  the  biuret  test.  Myers  attempted  to  determine  whether  or  not 
the  precipitin  is  used  up  in  the  formation  of  the  precipitum  and  on 
this  point  he  makes  the  following  statement :  "  A  mixture  of  the 
precipitins  of  bullock's  globulin  and  egg  albumin  was  added  to  a  so- 
lution of  egg  albumin.  After  standing  at  37°  for  fifteen  hours  the 
mixture  was  centrifugalized  and  the  clear  fluid  was  tested  for  the 
presence  of  the  precipitins.  It  was  found  to  give  a  precipitum  with 
bullock's  globulin  but  to  give  none  with  egg  albumin.  At  the  same 
time  a  control  experiment  was  made  with  a  mixture  of  the  precipi- 
tins of  egg  albumin  and  bullock's  globulin  with  bullock's  globulin. 
In  this  case  the  clear  fluid  precipitated  with  egg  albumin  but  not 
with  bullock's  globulin.  Experiments  of  a  similar  kind  with  the 
precipitins  of  egg  albumin  and  sheep's  globulin  were  made,  and  in 
this  case  also  the  one  or  other  of  the  precipitins  disappeared.  From 
these  experiments  it  is  concluded  that  the  precipitins  are  used  up  in 
the  course  of  their  action,  and  bearing  in  mind  their  specificity,  this 
is  strong  evidence  that  the  action  of  these  bodies  is  chemical." 

Uhlenhuth  ^  broke  freshly  laid  eggs  in  sterilized  beakers,  diluted 
with  physiological  salt  solution,  stirred  with  sterilized  rods,  and  thus 
prepared  a  solution  which  could  be  easily  injected  intra-peritoneally 
into  rabbits.  In  this  way  he  introduced  into  each  animal  at  each 
time  the  whites  of  from  two  to  three  eggs,  and  notwithstanding  the 
large  volume  of  the  fluid,  which  frequently  measured  100  c.c,  it  was 
well  borne.  After  this  operation  had  been  repeated  several  times 
the  rabbits  furnished  ovasera  which  proved  to  be  most  delicate  re- 
agents in  testing  for  egg  albumin,  inasmuch  as  a  few  drops  of  such  a 
serum  added  to  egg  albumin  diluted  with  one  hundred  thousand  parts 
of  water,  give  a  distinct  cloudiness,  while  nitric  acid  and  acetic  acid 
and  ferrocyanide  of  potassium  fail  to  indicate  the  presence  of  albumin 
when  the  solution  is  diluted  only  1:1000.  Moreover,  this  reaction 
1  Deutsche  med.  Wochenschrift,  1900. 


PRECIPITINS.  117 

is  specific  and  these  ovasera,  which  furnish  such  delicate  reagents  for 
testing  for  egg  albumin,  do  not  give  any  precipitate  with  alkaline 
albuminates,  peptons,  casein,  or  blood  serum.  However,  it  was 
found  that  ovasera  obtained  from  animals  immunized  to  the  albumin 
of  hen's  eggs  do  react  with  the  albumin  of  pigeon's  eggs.  Uhlenhuth 
obtained  feebly  active  ovasera  by  prolonged  and  excessive  feeding  of 
rabbits  with  egg  albumin  by  the  mouth.  However,  the  serum  thus 
obtained  was  only  slightly  active  and  it  is  probable  that  the  small 
amount  of  precipitin  in  this  serum  was  due  to  traces  of  egg  albumin 
absorbed  unchanged  through  the  walls  of  the  stomach  or  intestines. 
Ovasera  may  be  heated  for  one  hour  at  60"  without  injury  to  their 
precipitins. 

Leclainche  and  Vallee  ^  injected  albuminous  urine  intravenously 
into  i^abbits.  They  used  in  this  way  20  c.c.  at  a  time  and  repeated 
at  intervals  until  each  animal  received  from  150  to  200  c.c.  The 
serum  of  animals  thus  treated  furnishes  a  very  delicate  test  for 
albuminous  urine,  giving  an  immediate  precipitation.  Urinsera 
do  not  precipitate  non-albuminous  urine.  Moreover,  if  the  ani- 
mal has  been  rendered  immime  with  a  urine  containing  serum 
globulin,  the  serum  from  this  animal  does  not  precipitate  urine  con- 
taining serum  albumin,  and  vice  versa.  Urinsera  also  precipitate 
pleuritic  and  other  exudates,  thus  showing  that  the  proteids  of  al- 
buminous urine  and  of  these  exudates  are  identical.  There  is  some 
contradiction  in  the  statements  concerning  the  action  of  urinsera  on 
the  blood  serum  of  man.  This  point  needs  more  extended  observa- 
tion. Mertens  ^  immunized  a  rabbit  with  placental  blood  serum  and 
found  that  the  serum  obtained  from  this  animal  produced  a  precipi- 
tum  in  human  blood  serum  and  in  albuminous  urine  from  man,  but 
was  without  effect  upon  the  blood  serum  of  normal  rabbits  or  upon 
the  albuminous  urine  of  a  rabbit  whose  kidneys  had  been  injured  by 
the  administration  of  cantharides. 

Zuelzer^  treated  rabbits  ^vith  from  5  to  10  c.c.  of  albuminous 
urine  (containing  from  1  to  9  per  m.  of  albumin)  at  intervals  of  from 
one  to  three  days  for  two  weeks,  obtaining  from  the  animals  thus 
treated  a  serum  which  precipitated  both  albuminous  urine  and  the 
blood  of  man. 

Tchistovitch  *  rendered  animals  immune  to  eel  serum  which  is 
toxic,  and  obtained  from  the  animals  thus  immunized  a  serum  which 
produced  a  precipitum  in  tlie  eel  serum.  His  conclusions  are  as 
follows  : 

] .  Rabbits,  dogs,  goats,  and  pigeons  are  easily  immunized  against 
the  serum  of  the  eel ;  it  is  infinitely  more  difficult  to  immunize 
guinea-pigs. 

*  Oomptes  Bendus  de  la  Society  de  Biologie,  1901. 
^Deutsche  med.  Wochenschrift,  1901. 

'  Deutsche  med.  Wochenschrift,  1901. 

*  Annates  de  V  InstUut  Pasteur,  13. 


118  THE  SPECIFIC  PRECIPITINS. 

2.  There  appears  in  the  blood  of  the  immunized  animals  an  anti- 
toxin which  in  vitro  neutralizes  the  solvent  action  of  the  toxin  on 
the  red  blood  corpuscles  of  the  rabbit,  and  which,  when  injected  into 
the  blood,  prevents  the  action  of  the  toxin. 

3.  This  antitoxin  appears  in  the  blood  promptly,  even  after  from 
two  to  four  injections,  and  in  the  rabbit  it  is  at  this  time  at  its 
maximum  strength ;  in  general,  its  antitoxic  value  is  not  great. 

4.  In  the  course  of  a  prolonged  immunization,  the  strength  of  the 
antitoxin  progressively  diminishes  while  the  resistance  of  the  rabbit 
against  the  toxin  increases. 

5.  The  red  blood  corpuscles  of  immunized  rabbits  are  less  soluble 
in  the  serum  of  the  eel  than  are  the  corpuscles  of  non-treated 
animals. 

6.  The  resistance  of  the  corpuscles  is  not  in  proportion  to  the 
amount  of  antitoxin  present  in  the  blood  of  the  immunized  animal ; 
on  the  contrary,  one  observes  a  certain  antagonism  between  the  re- 
sistance of  the  corpuscles  and  the  strength  of  the  antitoxin,  and  in 
instances  in  which  the  latter  is  of  relatively  great  strength  the  solu- 
bility of  the  corpuscles  may  be  slightly  augmented. 

7.  The  injection  of  eel  serum  into  the  blood  is  followed  by  a  nota- 
ble diminution  in  the  number  of  leucocytes  in  an  untreated  animal 
and  an  augmentation  or  a  slight  diminution  in  immunized  animals. 
In  a  mixture  of  eel  serum  with  an  antitoxin  obtained  from  an  im- 
munized animal  one  observes  a  cloudiness  and  a  precipitation  similar 
to  that  reported  by  Kraus  in  filtered  bacterial  cultures.  This  pre- 
cipitate is  insoluble  in  water,  in  neutral  salts,  and  in  alkaline  car- 
bonates, but  is  easily  soluble  in  alkalis  and  acids.  Its  formation 
resembles  the  coagulation  of  a  substance  dissolved  in  toxic  or  anti- 
toxic serum. 

8.  The  volume  of  this  precipitate  is  ordinarily  in  proportion  to 
the  strength  of  the  antitoxin  ;  its  formation  is  retarded  by  heating 
the  antitoxin  to  70°  for  one-half  hour;  and  heating  the  eel  serum  to 
80°  renders  it  incoagulable  by  the  antitoxin. 

9.  The  coagulation  is  not  directly  due  to  the  antitoxin  for  one  may 
obtain  antitoxic  sera  which  do  not  give  a  coagulum. 

10.  Antitoxic  sera  acquire  very  promptly  the  property  of  agglu- 
tinating the  red  corpuscles  of  the  animals  whose  blood  or  serum  is 
injected. 

11.  The  power  of  agglutinating  the  red  blood  corpuscles  does  not 
correspond  with  the  coagulating  power  and  the  former  may  be  pro- 
nounced in  a  serum  which  does  not  coagulate  its  homologous  toxin. 

12.  The  agglutination  of  red  blood  corpuscles  is  not  caused  by  the 
coagulation  of  substances  dissolved  in  the  liquid  portion. 

Bordet  ^  injected  the  defibrinated  blood  of  the  chicken  into  rabbits 
and  obtained  from  the  latter  a  serum  which  produced  a  precipitum 

^  Annales  de  Plnatitut  Pasteur,  13. 


PRECIPITINS.  119 

in  chickens'  blood,  also  agglutinized  and  dissolved  the  corpus- 
cles. Uhlenhuth  ^  treated  rabbits  intraperitoneally  with  dilutions  of 
chickens'  blood,  and  obtained  from  the  former  animals  a  serum  which 
gave  a  precipitum  with  chickens'  blood,  but  had  no  such  action  on 
the  blood  of  the  horse,  donkey,  cow,  sheep  or  pigeon ;  nor  did  it 
produce  any  precipitum  in  solutions  of  egg  albumin,  nor  in  the  serum 
of  normal  rabbits. 

Wolffs  allowed  chickens'  blood  to  flow  into  four  times  its  volume 
of  a  one  per  cent,  solution  of  salt  and  separated  the  corpuscles  from 
the  plasma  and  injected  both  of  these  separately  into  two  sets  of 
rabbits.  From  four  to  six  injections  at  intervals  of  four  to  five  days 
were  made  and  only  those  animals  treated  with  the  blood  plasma 
yielded  a  precipitum  with  chicken  blood  ;  those  treated  with  the  cor- 
puscles had  no  such  effect.  Other  rabbits  treated  in  the  same  man- 
ner with  dog's  blood  corpuscles  and  plasma  gave  similar  results,  the 
precipitins  being  found  only  in  those  animals  which  had  been  treated 
with  the  fluid  portion  of  the  blood. 

Uhlenhuth  ^  treated  rabbits  intraperitoneally  at  intervals  of  from 
six  to  eight  days  with  10  c.c.  of  defibrinated  blood  at  each  injection. 
After  the  fifth  injection  the  serum  from  the  treated  animals  was  ob- 
tained and  used  for  testing  blood  from  various  sources.  The  blood  to 
be  tested  was  diluted  with  tap  water  until  it  was  reduced  to  a  feebly 
red  color  (1 :100).  This  diluted  blood  was  freed  from  stroma  either  by 
filtration  or  by  decantation,  and  two  c.c.  of  it  was  placed  in  test-tubes 
and  diluted  with  an  equal  volume  of  double  physiological  salt  solu- 
tion (1.6  per  cent.).  It  is  important  that  blood  should  be  diluted 
with  physiological  salt  solution  and  not  wholly  with  water,  which  is 
likely  to  cloud  certain  bloods  when  diluted.  Blood  thus  prepared 
was  obtained  from  the  ox,  horse,  donkey,  pig,  sheep,  dog,  cat,  deer, 
hare,  guinea-pig,  rat,  mouse,  rabbit,  chicken,  goose,  turkey,  pigeon  and 
man.  To  tubes  containing  diluted  blood  from  each  of  the  above- 
mentioned  animals,  from  six  to  eight  drops  of  the  serum  of  the  rab- 
bit which  had  been  treated  with  ox  blood  was  added.  The  ox  blood 
was  immediately  clouded,  while  all  others  remained  perfectly  clear. 
The  clouding  in  the  first  mentioned  grew  in  intensity  and  finally 
formed  floccules  which  gradually  subsided.  It  will  be  seen  that 
by  this  test  ox  blood  was  easily  distinguished  from  all  others  with 
which  it  was  compared.  Uhlenhuth  was  also  able  to  distinguish 
ox  blood  from  that  of  man  or  the  horse,  the  three  kinds  having  been 
obtained  from  stains  four  weeks  old. 

Stern  *  treated  animals  subcutaneously  with  human  blood,  employ- 
ing from  five  to  ten  c.c.  at  each  injection  with  intervals  of  two  or 
more  days.     After  from  two  to  three  weeks,  he  obtained  from  the 

1  Deutsche  Tried.  Wockenschrift,  1900. 
^Annales  de  V  Itistitut  Pasteur,  14. 
'  Deutsche  med.  Wochenschrift,  1901. 
*  Deutsche  med.  Wochenschrift,  1901. 


120  THE  SPEGIFIG  PRECIPITINS. 

rabbits  a  serum  which  precipitated  the  blood  serum  obtained  from 
man,  also  albuminous  urine  from  man,  and  did  not  precipitate  the 
blood  serum  of  the  horse,  sheep  or  ox.  He  states,  however,  that 
the  reaction  is  not  wholly  a  specific  one,  inasmuch  as  the  serum  ob- 
tained in  this  case  gave  marked  cloudiness  with  blood  sera  obtained 
from  three  different  species  of  ape. 

Wassermann  and  Schiitze  ^  tested  serum  obtained  from  rabbits 
treated  with  human  blood  on  23  kinds  of  blood,  and  found  that 
none  of  these  reacted  except  blood  from  man  and  from  a  baboon ; 
however,  the  reaction  with  the  blood  of  the  baboon  was  not  nearly 
so  marked  as  that  with  the  blood  of  man. 

Dieudoun§  ^  treated  rabbits  with  human  blood  serum,  albuminous 
urine,  and  pleuritic  exudate.  The  rabbits  treated  with  human  blood 
serum  furnished  a  serum  which  gave  a  precipitum  with  human  blood 
and  had  no  effect  on  the  blood  of  the  rabbit,  the  guinea-pig,  the 
pigeon  or  the  goose.  The  animals  treated  with  albuminous  urine 
gave  a  precipitum  in  the  albuminous  urine  of  man  and  had  no  effect 
upon  the  normal  urine  of  man  or  that  of  the  rabbit.  The  serum  ob- 
tained from  animals  treated  with  human  pleuritic  exudate  gave  no 
reaction  with  a  similar  exudate  obtained  from  a  guinea-pig.  The 
serum  obtained  from  the  rabbits  treated  with  human  blood  serum 
gave  a  precipitum  not  only  with  human  blood,  but  also  with  human 
albuminous  urine  and  the  pleuritic  and  peritoneal  exudates  obtained 
from  man. 

Nuttall  ^  obtained  rabbit  sera  by  treating  these  animals  with  the 
blood  of  man,  dog,  sheep,  ox  and  horse.  These  sera  were  tested 
against  thirty-six  kinds  of  blood.  The  serum  of  rabbits  treated  with 
dog  serum  gave  negative  results  in  all  cases  except  with  the  blood  of 
the  dog.  The  serum  of  rabbits  treated  with  sheep  serum  produced 
a  marked  precipitum  with  sheep's  blood,  and  a  distinct,  but  less 
marked,  reaction  with  the  blood  of  the  gazelle  and  axis  deer.  All  the 
other  sera  and  bloods  remained  perfectly  clear,  excepting  that  of  the 
ox,  squirrel  and  swan,  in  which  there  was  very  slight  cloudiness. 
The  serum  of  the  rabbits  treated  with  ox  serum  produced  a  marked 
precipitation  only  in  ox  serum  dilutions  or  dried  ox  blood  solutions, 
but  gave  a  distinct  reaction  with  the  blood  of  the  gazelle  and  axis 
deer.  All  the  other  bloods  gave  a  negative  reaction,  a  slight  cloud- 
ing only  being  produced  in  the  blood  serum  of  the  sheep,  gnu, 
squirrel  and  swan.  The  serum  of  rabbits  treated  with  horse  serum 
produced  a  precipitum  only  in  dilutions  of  the  horse  blood  or  serum, 
not  even  clouding  other  bloods.  The  serum  of  the  rabbits  treated 
with  human  blood  serum  and  pleuritic  exudate  produced  a  marked 
precipitum  only  in  human  blood  solutions,  but  the  blood  of  four  spe- 

^  Berliner  klin.  Wochenschrift,  1901. 
*  Munchener  med.  Wochentchrift,  1901. 
'Journal  of  Hygiene,  1. 


PRECIPITINS.  121 

cies  of  monkeys  gave  a  slight  but  distinct  reaction  and  a  very  faint 
clouding  appeared  in  the  solutions  of  the  bloods  of  the  horse,  ox  and 
sheep,  all  other  bloods  remaining  perfectly  clear.  "  The  test  gave 
positive  results  when  made  with  diluted  human  serum,  pleuritic  ex- 
udation, both  fresh  and  purulent,  blood  and  serum  which  had  been 
dried  on  filter  paper  and  on  glass  plates,  with  blood  which  had 
undergone  putrefaction  for  two  months,  with  the  blood  of  several 
persons  who  had  cut  themselves  (blood  collected  on  filter  paper),  with 
the  serum  from  a  blister  on  the  foot  following  upon  a  long  walk,  and 
with  the  serum  from  a  blister  following  a  burn  on  the  hand.  Both 
nasal  and  lachrymal  secretion  gave  a  slight  but  decided  reaction.  A 
faint  clouding  was  produced  in  normal  urine.  That  the  precipitum 
formed  in  putrid  blood  dilution  was  specific  was  proved  by  adding 
the  anti-sera  of  rabbits  treated  with  ox,  sheep  and  dog  serum  to  the 
blood  dilution,  no  reaction  resulting." 

Nuttall  concludes  his  investigations  as  follows :  "  (1)  The  investi- 
gations we  have  made  confirm  and  extend  the  observations  of  others 
with  regard  to  the  formation  of  specific  precipitins  in  the  blood  serum 
of  animals  treated  with  various  sera.  (2)  These  precipitins  are 
specific,  although  they  may  produce  a  slight  reaction  with  the  sera 
of  allied  animals.  (3)  .^The  substance  in  serum  which  brings  about 
the  formation  of  a  precipitin,  as  also  the  precipitin  itself,  are  re- 
markably stable  bodies.  (4)  The  new  test  can  be  successfully 
applied  to  a  blood  which  has  been  mixed  with  those  of  several  other 
animals.  (5)  We  have  in  this  test  the  most  delicate  means  hitherto 
discovered  of  detecting  and  testing  bloods,  and  consequently  we 
may  hope  that  it  will  be  put  to  forensic  use." 


CHAPTER  VIL 

THE   LYSINS. 

One  of  the  most  important  contributions  to  the  science  of  bacteri- 
ology made  during  the  last  decade  of  the  nineteenth  century  was  the 
discovery  of  what  is  known  as  Pfeiffer's  phenomenon.  In  his  ex- 
periments, R.  Pfeiffer  discovered  that  if  cholera  bacteria  are  placed 
in  the  peritoneal  cavity  of  a  guinea-pig  which  has  been  immunized 
to  cholera,  the  bacterial  cells  are  dissolved  by  the  peritoneal  fluid. 
If  such  an  injection  be  made  and  portions  of  the  bacilli  be  removed 
with  capillary  glass  tubes  every  five  minutes,  it  can  be  plainly  seen, 
under  the  microscope,  that  the  bacterial  cells  are  undergoing  solu- 
tion in  the  surrounding  fluid  just  as  lumps  of  salt  dissolve  in  water. 
The  bacilli  lose  their  motility,  swell  up,  and  then  break  into  small 
granules  which  gradually  melt  away  into  the  fluid.  Extended  in- 
vestigation showed  that  this  reaction  is  specific  and  the  same  phe- 
nomenon may  be  observed  in  animals  which  have  been  immunized 
to  the  typhoid  bacillus  or  other  pathogenic  microorganisms,  and  by 
this  means  the  cholera  bacillus  may  be  diflFerentiated  from  other 
vibrios.  Moreover,  when  a  mixed  culture  is  subjected  to  this  test, 
the  peritoneal  fluid  dissolves  the  bacteria  of  the  species  against 
which  the  animal  has  been  immunized  and  leaves  all  other  bacteria 
untouched.  Pfeiffer  found  that  it  was  not  necessary  to  use  an  im- 
munized animal,  but  that  the  same  result  can  be  obtained  by  placing 
a  small  quantity  of  a  cholera  culture  mixed  with  the  serum  of  a 
guinea-pig  which  has  been  immunized  to  cholera,  in  the  abdominal 
cavity  of  a  normal  guinea-pig.  Later,  Metschnikoff  ^  discovered 
that  Pfeiffer's  phenomenon,  which  is  also  known  as  bacteriolysis, 
takes  place  in  vitro,  when  to  a  mixture  of  cholera  serum  and  the 
cholera  culture  there  be  added  a  small  quantity  of  the  peritoneal 
exudate  from  a  normal  guinea-pig,  and  Bordet  added  the  observa- 
tion that  cholera  serum  in  and  of  itself  suffices  to  induce  bacteri- 
olysis in  vitro  when  it  is  perfectly  fresh.  On  long  standing  it  be- 
comes inactive,  but  its  activity  even  then  may  be  restored  by  the 
addition  of  a  small  quantity  of  normal  serum.  Pfeiffer  repeated 
Bordet's  experiments,  and  confirmed  them  in  a  modified  way,  inas- 
much as  Pfeiffer  found  that  not  every  fresh  blood  serum  had  the 
same  effect,  and  that  the  process  of  bacteriolysis  as  observed  in  vitro 
is  not  comparable  in  intensity  with  that  observed  in  the  animal  body. 

'  AnncUee  dePInstitut  Pasteur,  1895. 
122 


THE  LYSINS.  123 

With  these  facts  at  his  command  Pfeiflfer  ^  formulated  a  theory  of 
bacteriolysis,  the  chief  points  of  which  may  be  stated  as  follows : 
The  immunizing  substance  in  cholera  serum  has  but  feeble  action  and 
is  only  the  antecedent  of  a  substance  formed  in  the  peritoneum  of 
the  guinea-pig  which  has  a  specific  solvent  action  on  the  vibrios. 
The  immunizing  substance  in  the  serum  is  a  stable,  relatively  inac- 
tive body  which  bears  a  relation  to  the  specific  bacteriolytic  substance 
formed  in  the  peritoneum  similar  to  that  which  glycogen  bears  to 
grape-sugar.  In  case  of  need,  the  inactive  substance  in  the  serum  is 
transformed  through  the  active  agency  of  the  cells  of  the  body  into 
the  specific  bacteriolytic  form.  This  change  can  be  brought  about 
by  the  addition  of  suitable  normal  serum.  In  the  added  serum  there 
is  a  "something"  (Etwas)  present  in  small  amount  which  is  able  to 
transform  the  relatively  inactive  substance  into  the  active  form,  but 
which  in  vitro  is  soon  used  up,  while  in  the  animal  body  this  active 
principle  continues  to  be  secreted  by  the  cells  of  the  body  as  long  as 
they  are  stimulated  by  the  presence  of  the  cholera  bacteria.  The 
active  principle  is  a  ferment  and  bacteriolysis  is  a  fermentative  proc- 
ess in  which  specific  ferments  act  only  on  certain  cells,  just  as  certain 
yeasts  act  only  on  sugars  of  certain  definite  constitution. 

The  question  of  the  identity  of  bacteriolytic  and  agglutinating  sub- 
stances in  the  sera  of  immunized  animals  became  prominent,  but 
PfeifFer  and  Kolle^  soon  discovered  an  immune  serum  which  was 
strongly  bacteriolytic  but  without  agglutinating  action ;  while  on  the 
other  hand,  Friinkel  and  Otto  ^  found  that  the  blood  serum  of  pup- 
pies fed  upon  typhoid  cultures  had  a  marked  agglutinating  effect,  but 
was  without  bacteriolytic  properties.  Moreover,  Widal  and  Si  card  * 
found  that  the  blood  serum  of  frogs  inoculated  with  typhoid  bacilli 
agglutinated  these  microorganisms  but  had  no  bacteriolytic  action  on 
them,  and,  indeed,  the  typhoid  bacilli  may  retain  both  life  and  viru- 
lence after  long  residence  in  the  lymph  sacs  of  such  frogs. 

Bordet®  treated  guinea-pigs  at  intervals  with  injections  of  the  de- 
fibrinated  blood  of  rabbits  and  obtained  from  the  former  animals  a 
serum  which  in  vitro  dissolved  the  blood  corpuscles  of  the  latter 
with  great  intensity,  while  the  serum  of  normal  guinea-pigs  was 
found  to  be  without  solvent  action  upon  the  corpuscles  of  the  rabbit. 
In  these  experiments  it  was  observed  that  the  serum  first  aggluti- 
nated and  then  dissolved  the  erythrocytes ;  but  more  extended 
investigation  showed  that  agglutination  does  not  always  precede  so- 
lution. Moreover,  Bordet  demonstrated  that  even  in  this  case  ag- 
glutination and  hemolysis  are  not  identical  or  due  to  the  same  sub- 
stances, inasmuch  as  he  found  that  a  temperature  of  55°  deprived 

'  Deutsche  med.  Wochenschrift,  1896. 
2  Centralblatt  f.  Bakieriologie,  20. 
'  Munchener  med.  Wochenschrift,  1894. 
*  Compt.  Bend.  Soc.  de  Biol.,  11,  XI. 
^Annates  de  V Institut  Pasteur,  12. 


124  THE  LYSINS. 

the  guinea-pig  serum  of  its  hemolytic  action,  but  had  no  effect  upon 
its  agglutinating  properties.  The  serum  rendered  inactive,  so  far  as 
its  hemolytic  properties  are  concerned,  by  heating,  was  found  to  re- 
cover these  properties  on  the  addition  of  certain  amounts  of  the 
serum  of  either  normal  guinea-pigs  or  rabbits.  The  active  serum 
was  found  to  be  without  effect  upon  the  corpuscles  of  the  guinea-pig 
itself  and  upon  those  of  the  pigeon,  but  with  slight  action  on  the  cor- 
puscles of  the  rat  and  the  mouse.  The  active  serum  from  the 
guinea-pig  was  found  to  be  powerfully  toxic  when  injected  intra- 
venously into  the  rabbit.  These  experiments  made  by  Bordet  dem- 
onstrated the  identity  of  the  processes  of  bacteriolysis  and  hemolysis  ; 
and  most  of  the  subsequent  investigation  has  been  confined 'to  a  study 
of  the  phenomenon  as  observed  in  the  solution  of  blood  corpuscles. 

Ehrlich  and  Morgenroth  ^  treated  a  goat  at  irregular  intervals  for 
eight  months  with  subcutaneous  injections  of  defibrinated  sheep's 
blood  diluted  with  0.85  per  cent,  salt  solution,  until  the  mixture  con- 
tained only  five  per  cent,  of  blood.  The  serum  of  this  goat  rapidly 
dissolved  the  erythrocytes  of  the  sheep  in  vitro  ;  while  the  serum  of 
a  normal  goat  had  but  slight  solvent  action  on  the  blood  corpuscles 
of  the  sheep.  In  this  case  agglutination  did  not  precede  hemolysis. 
When  the  immune  serum  was  heated  for  half  an  hour  at  56°,  it  lost 
its  solvent  action,  but  on  the  addition  of  the  serum  of  a  normal  ani- 
mal its  activity  was  regained.  The  added  serum  was  active  when 
obtained  from  either  the  goat  or  the  sheep,  but  less  so  from  the  latter. 
Normal  serum  was  found  to  soon  lose  its  power  of  restoring  the 
activity  of  heated  immune  serum  and  was  effective  only  when  freshly 
obtained  from  the  animal.  These  investigators  agreed  with  Pfeiffer 
that  at  least  two  substances  must  be  present  in  the  blood  serum  in 
order  to  induce  bacteriolysis  or  hemolysis.  For  the  first,  which  is 
thermo-stable,  they  retain  the  name  proposed  by  Pfeiffer,  of  "  immune 
body  "  ;  while  for  the  second,  which  is  thermo-labile,  they  substitute 
for  the  "  something  "  of  Pfeiffer  the  term  "  addiment."  It  will  be 
understood  that  the  thermo-stable  or  immune-body  is  unaltered  by  a 
heat  of  56°,  while  the  thermo-labile  substance,  or  addiment,  is  de- 
stroyed at  this  temperature,  but  is  also  contained  in  normal  serum ; 
and  this  explains  why  the  immune  serum  after  being  rendered  in- 
active by  heat  is  regenerated  by  the  addition  of  a  small  amount  of 
normal  serum.  It  was  at  first  supposed  by  Ehrlich  that  the  immune 
body  is  a  substance  which  is  brought  into  existence  during  the  proc- 
ess of  immunizing  the  animal,  while  the  addiment  was  supposed  to 
be  present  normally  in  the  blood.  Ehrlich's  theory  provided  for  a 
combination  between  the  immune  body  and  the  blood  corpuscles ; 
however,  this  does  not  lead  to  solution  of  the  corpuscle  until  the 
substance  addiment  is  brought  into  the  compound.  The  following 
experiments  furnish  the  basis  of  this  theory :  Tubes  each  containing 

*  Berliner  klin.  Wochenschrift,  1899. 


THE  LYSINS.  120 

four  c.c.  of  the  five  per  cent,  dilution  of  sheep's  blood  were  treated 
with  from  one  to  one  and  three-tenths  c.c.  each  of  the  serum  of  the 
goat  rendered  inactive  by  heat.  The  mixture  was  allowed  to  remain 
for  fifteen  minutes  at  40°  and  then  centrifuged.  The  supernatant 
clear  fluid  was  removed  and  treated  with  0.2  c.c.  of  normal  sheep's 
blood  and  then  0.8  c.c.  of  the  serum  from  a  normal  goat  added. 
This  mixture  was  allowed  to  stand  for  two  hours  at  37°,  when  it 
was  found  that  no  hemolysis  had  occurred.  Next,  the  sediment 
which  had  formed  in  the  centrifuge  was  placed  in  four  c.c.  of  physio- 
logical salt  solution  and  0.8  c.c.  of  the  blood  of  a  normal  goat  added. 
When  this  mixture  was  allowed  to  stand  at  37°  for  two  hours,  it  was 
found  that  all  the  blood  corpuscles  were  dissolved.  This  experiment 
can  be  explained  only  by  supposing  that  the  immune  body  in  the 
heated  serum  combined  with  the  corpuscles  and  subsided  with  them 
in  the  centrifuge,  leaving  the  supernatant  fluid  without  the  immune 
body,  and  therefore  the  addition  of  the  addiment  to  the  supernatant 
fluid  did  not  induce  hemolysis.  On  the  other  hand,  the  presence  of 
the  immune  body  in  the  sediment  was  shown  when  hemolysis  oc- 
curred after  distributing  the  sediment  in  saline  solution  and  adding 
a  small  quantity  of  normal  blood  serum. 

It  was  shown  that  the  combination  between  the  corpuscles  and  the 
immune  body  may  take  place  at  low  as  well  as  at  higher  temperatures, 
while  the  addiment  does  not  enter  into  the  compound  at  low  temper- 
ature. When  five  c.c.  of  the  five  per  cent,  sheep's  blood  dilution  was 
treated  with  from  1  to  1.3  c.c.  of  inactive  serum  and  0.5  c.c.  of  the 
serum  of  a  normal  goat  added,  and  this  mixture  kept  at  37°,  com- 
plete hemolysis  occurred  within  two  hours.  On  the  other  hand,  when 
the  same  mixture  was  kept  at  from  0°  to  3°  there  was  no  hemolysis, 
but  at  this  temperature  it  was  shown  by  a  repetition  of  the  experi- 
ment already  detailed  that  the  immune  body  was  combined  in  the 
sediment  with  the  corpuscles,  while  the  addiment  remained  in  the 
supernatant  fluid. 

It  was  also  shown  that  at  high  temperatures  the  combination  be- 
tween the  corpuscles  and  the  immune  body  occurs  before  the  addi- 
ment enters  into  the  compound.  Tubes  containing  blood  corpuscles, 
inactive  serum,  and  fresh  goat  serum,  were  placed  in  a  water-bath  at 
40°  and  the  time  before  visible  solution  of  the  corpuscles  took  place 
was  noted.  It  was  found  that  no  evident  hemolysis  occurred  within 
ten  minutes ;  therefore  the  tubes  which  had  been  kept  at  40°  for  ten 
minutes  were  centrifuged.  When  the  sediment  in  the  centrifuge 
was  distributed  through  physiological  salt  solution  slight  hemolysis 
occurred,  but  complete  hemolysis  resulted  only  after  the  addition  of 
normal  serum  to  this  mixture.  This  experiment  demonstrated  that 
all  of  the  immune  body  had  combined  with  the  corpuscles  and  had 
subsided  with  them,  while  the  greater  part  of  the  addiment  remained 
in  the  supernatant  fluid.     The  affinity  of  the  immune  body  for  the 


126  THE  LYSINS. 

blood  corpuscles  is  great,  and  leads  to  speedy  combination  between 
the  two  both  at  high  and  low  temperatures ;  while  the  affinity  of  the 
immune  body  for  the  addiment  is  slight  and  leads  to  combination 
tardily  and  only  at  relatively  high  temperatures.  The  immune  body 
is  supposed  to  be  possessed  of  two  haptophorous  groups,  one  of  which 
has  great  chemical  energy,  and  is  that  by  which  the  immune  body 
combines  with  the  corpuscle ;  while  the  other  haptophorous  group 
possesses  less  chemical  energy  and  is  that  by  means  of  which  combi- 
nation with  the  addiment  is  eifected.  In  the  first  statement  of  his 
theory,  Ehrlich  believed  that  the  addiment  is  a  ferment  which  digests 
and  dissolves  bacteria  and  blood  corpuscles.  He  stated :  "  It  is 
possible,  yes,  probable,  that  there  are  in  the  blood  only  a  few,  prob- 
ably only  a  single  body,  which  has  digestive  properties ;  while,  on 
the  other  hand,  there  must  be  innumerable,  different,  specific  immune 
bodies,  as  Gruber  and  others  have  supposed.  One  may  presume 
therefore  that  in  the  different  immune  bodies  only  the  group  which 
combines  with  the  immunizing  substance  differs,  while  the  group 
which  combines  with  the  digestive  ferment  is  the  same  in  all." 

In  a  second  series  of  experiments,  Ehrlich  and  Morgenroth  ^  im- 
munized two  goats  with  gradually  increased  quantities  of  defibrinated 
sheep's  blood  injected  subcutaneously.  From  these  animals  there 
were  obtained  highly  active  sera.  The  serum  of  the  first  animal  in 
quantities  of  from  0.2  to  0.3  c.c.  completely  dissolved  five  c.c.  of  the 
dilution  of  sheep's  blood,  while  from  0.03  to  0.07  c.c.  had  a  marked 
effect.  The  serum  of  the  second  goat  in  quantities  of  from  0.15  to 
0.2  c.c.  completely  dissolved  five  c.c.  of  the  dilution  of  sheep's  blood. 
It  is  stated  that  the  serum  of  goat  II  before  the  process  of  immuniz- 
ing had  a  feebly  solvent  action  on  sheep's  blood,  inasmuch  as  four  c.c. 
of  the  serum  partially  dissolved  five  c.c.  of  the  five  per  cent,  dilution 
of  sheep's  blood.  Heating  to  57°  for  half  an  hour  destroyed  this 
action,  also  the  solvent  action  of  the  same  serum  on  rabbit's  and 
guinea-pig's  blood.  It  was  shown  that  the  sera  of  both  these  animals 
contained  the  immune  body  which  combines  with  the  corpuscles  at 
0°.  Mixtures  of  these  sera  and  sheep's  blood  were  allowed  to  stand 
for  twenty-four  hours  at  0°  and  then  the  corpuscles  were  separated 
in  the  centrifuge.  When  the  sediment  was  shaken  up  with  physio- 
logical salt  solution  there  was  no  hemolysis  until  the  addiment  in 
the  form  of  normal  goat  serum  was  added.  At  a  temperature  of  20° 
continued  for  eight  minutes,  both  components,  the  immune  body  and 
the  addiment,  combined  with  the  corpuscles  and  a  sediment  obteined 
from  this  mixture  and  distributed  in  physiological  salt  solution  and 
kept  at  37°  resulted  in  complete  hemolysis.  The  sera  obtained  from 
these  animals  differed  from  that  obtained  from  the  goat  in  the  first 
series  in  being  more  intense  in  action  and  in  the  fact  that  when  heated 
to  56°  for  three-quarters  of  an  hour  there  was  scarcely  any  reduction 

'  Berliner  klin.  Wochenschri/t,  1899,  481. 


THE  LYSINS.  127 

in  their  hemolytic  action  on  sheep's  blood,  while  their  action  on  the 
blood  corpuscles  of  the  guinea-pig  and  the  rabbit  was  completely  de- 
stroyed. Indeed,  heating  for  three  hours  at  56°,  or  heating  the  serum 
diluted  with  an  equal  volume  of  water  for  one-half  hour  at  65°,  had 
no  effect  upon  the  hemolytic  action  on  sheep's  blood.  This  indicated 
that  the  addiment  in  these  sera  differs  from  that  observed  in  the 
goat  experimented  upon  in  the  first  series,  and  the  fact  that  heating 
to  56°  destroyed  the  action  of  these  sera  on  the  blood  of  the  guinea- 
pig  and  of  the  rabbit  suggested  that  they  contain  at  least  two  addi- 
ments,  one  of  which  is  necessary  to  the  hemolytic  action  on  the  blood 
of  the  guinea-pig  and  the  rabbit  and  is  destroyed  at  56°,  while  the 
other  is  that  by  virtue  of  which  the  hemolytic  action  on  sheep's  blood 
results  and  which  is  not  destroyed  at  a  temperature  of  56°.  In  other 
words,  there  must  be  in  these  sera  two  addiments  one  of  which  is 
thermo-stable,  while  the  other  is  thermo-labile. 

In  order  to  solve  the  problem  presented  by  this  new  discovery, 
Ehrlich  and  Morgenroth  determined  to  separate  the  two  components 
in  these  sera.  The  immune  body  was  easily  obtained  by  combining 
it  with  erythrocytes  at  0°  and  it  was  found  that  when  these  sera  were 
treated  with  10  per  cent,  of  their  volume  of  normal  hydrochloric  acid 
and  the  mixture  digested  for  from  thirty  to  forty-five  minutes  at  37° 
and  neutralized,  they  lost  their  hemolytic  effect  on  sheep's  blood. 
The  acid  destroyed  the  specific  addiment  in  these  sera,  but  had  no 
effect  upon  the  immune  body.  The  experiment  was  carried  out  as 
follows  :  To  five  c.c.  of  the  five  per  cent,  sheep's  blood  dilution  there 
was  added  0.15  c.c.  of  the  immune  serum  rendered  inactive  with  hy- 
drochloric acid  (it  having  been  previously  shown  that  this  amount  of 
active  serum  was  sufficient  to  produce  complete  hemolysis  in  five  c.c. 
of  the  blood  dilution).  After  the  mixture  had  stood  for  half  an  hour 
at  room  temperature,  it  was  centrifuged  and  separated  into  sediment 
and  supernatant  fluid.  To  the  sediment  there  was  added  two  c.c.  of 
normal  goat  serum  and  to  the  supernatant  fluid  there  was  added 
another  portion  of  the  diluted  sheep's  blood,  and  also  two  c.c.  of  nor- 
mal goat  serum.  When  thus  treated  the  corpuscles  in  the  sediment 
were  completely  dissolved,  while  the  supernatant  fluid  had  no  action 
upon  the  erythrocytes  which  had  been  added,  notwithstanding  the 
presence  in  it  of  the  addiment.  This  showed  that  all  of  the  immune 
body  was  contained  in  the  sediment.  However,  this  experiment  also 
showed  that  the  addiment  needed  to  render  the  immune  body  active 
is  present  in  normal  goat  serum  and  indicates  that  there  is  in  normal 
serum  a  thermo-stable  addiment ;  but  it  was  shown  that  the  thermo- 
stable addiment  was  not  present  in  the  sera  of  all  goats.  It  was 
concluded  that  in  the  serum  of  the  goat  used  in  the  first  experiment 
and  in  the  sera  of  the  goats  used  in  the  second  experiment  the  same 
immune  body  was  present,  but  that  the  serum  of  the  first  immunized 
goat  contained  only  thermo-labile  addiment,  while  those  of  the  two 


128  THE  LYSINS. 

goats  used  in  the  second  experiment  contained  both  thermo-labile  and 
thermo-stable  addiments. 

Another  experiment  showed  that  the  blood  serum  of  a  non-treated 
normal  animal  dissolved  the  erythrocytes  of  the  guinea-pig  and  that 
there  is  in  the  normal  serum  of  the  goat  a  substance  analogous  to  the 
immune  body  found  in  immunized  animals,  which  combines  with  the 
corpuscles  of  the  guinea-pig  at  0°.  It  is  evident  from  this  that  the 
term  "  immune  body "  is  not  appropriate  for  the  substance  which 
combines  with  the  erythrocytes  at  0°  and  which  is  one  of  the  hemo- 
lytic factors.  This  led  Ehrlich  to  substitute  for  the  term  "  immune 
body  "  the  designation  of  "  intermediary  body,"  ^  and  inasmuch  as 
he  had  demonstrated  that  there  might  be  different  addiments  in  the 
blood,  he  also  dropped  this  term  and  used  in  its  stead  "complex 
ment."  It  should  be  understood  that  the  "  immune  body,"  which 
we  will  hereafter  designate  as  "  intermediary  body,"  has  two  hap- 
tophorous  groups,  one  of  which  is  possessed  of  great  avidity,  and  it  is 
by  means  of  this  that  the  intermediary  body  combines  with  the  cor- 
puscles, and  its  great  avidity  is  shown  by  the  fact  that  this  combi- 
nation takes  place  even  at  0°.  The  other  haptophorous  group  of  the 
intermediary  body  is  possessed  of  less  avidity  and  it  is  by  means  of 
this  that  combination  between  the  intermediary  body  and  the  com- 
plement takes  place ;  and  on  account  of  the  slight  avidity  of  this 
haptophorous  group  this  combination  occurs  only  at  a  relatively  high 
temperature.  When  an  active  serum  is  kept  at  0°  it  contains  the 
intermediary  body  and  the  complement  both  in  a  free  state ;  now,  if 
susceptible  erythrocytes  be  added  to  this  serum  still  kept  at  0°,  the 
corpuscles  and  the  intermediary  body  combine,  while  the  complement 
remains  free ;  but  when  the  temperature  is  raised  to  37°  the  comple- 
ment enters  into  the  combination  by  attaching  itself  to  the  interme- 
diary body.  The  haptophorous  group  by  which  the  intermediary 
body  combines  with  the  corpuscle  is  sometimes  designated  as  hemo- 
tropic.  Of  course,  there  may  be  in  certain  sera  intermediary  bodies 
whose  hemotropic  groups  have  no  greater  avidity  than  their  comple- 
ment groups ;  or  there  may  be  complements  which  combine  with  in- 
termediary bodies  at  a  low  temperature.  It  may  happen  that  the 
intermediary  body  is  contained  in  the  serum  of  one  animal  while  the 
complement  may  be  furnished  by  the  blood  of  another  animal. 
This  has  been  shown  to  be  the  case  in  the  following  experiment : 
Dog  serum  dissolves  the  blood  corpuscles  of  the  guinea-pig  with 
great  energy.  If  the  dog  serum  be  heated  to  57°  it  loses  its  hemo- 
lytic action  on  guinea-pig  blood.  But  if  inactive  dog  serum  be 
added  to  five  c.c.  of  a  five  per  cent,  dilution  of  guinea-pig  blood  and 
two  c.c.  of  the  serum  of  a  normal  guinea-pig  be  added  to  this  mix- 
ture, complete  hemolysis  occurs.     This  is  explainable  only  on  tbfi 

'In  his  later  papers  Ehrlich  has  used  the  terms  "  immune  body "  and  "inter- 
mediary body ' '  interchangeably. 


THE  LYSINS.  129 

supposition  that  the  intermediary  body  exists  in  the  dog  serum,  while 
the  complement  is  furnished  by  the  serum  of  the  guinea-pig ;  in  this 
case  the  complement  is  furnished  by  the  serum  of  the  animal  whose 
corpuscles  are  dissolved.  It  is  probable  that  in  various  sera  both  the 
intermediary  bodies  and  the  complements  diflfer,  and  it  is  not  always 
possible  to  restore  the  hemolytic  action  of  a  serum,  which  has  been 
heated,  by  the  addition  of  a  complement.  For  instance,  eel  serum 
is  hemolytic  to  the  blood  of  most  mammals ;  it  loses  its  hemolytic 
action  when  heated  for  fifteen  minutes  to  54°,  and  so  far  no  method 
of  restoring  its  activity  has  been  discovered. 

In  a  third  communication  Ehrlich  and  Morgenroth  ^  carried  out 
their  immunization  experiments  in  a  wholly  different  way.  920  c.c. 
of  the  mixed  blood  from  three  goats  (Nos.  1,  2  and  3),  diluted  with 
750  c.c.  of  water,  was  injected  into  the  abdominal  cavity  of  a  large 
goat  [A)  at  one  time.  From  the  second  day  on  small  quantities  of 
the  blood  were  taken  from  this  animal  and  the  hemolytic  action  of 
the  serum  tested.  The  first  experiment  showed  that  this  serum 
possessed  slight  hemolytic  effect  on  the  corpuscles  of  other  goats, 
and  on  the  seventh  day  it  reached  its  maximum.  At  this  time  0.3 
c.c.  of  the  serum  completely  dissolved  the  corpuscles  in  one  c.c.  of  a 
five  per  cent,  dilution  of  goat  No.  4.  It  was  tested  upon  nine  goats 
and  it  was  found  that  the  susceptibility  of  the  erythrocytes  of  these 
animals  to  the  serum  varied  somewhat.  The  serum  of  the  treated 
animal  was  found  to  be  without  effect  upon  its  own  corpuscles. 
Ehrlich  suggests  that  the  hemolytic  action  of  the  blood  serum  of  one 
animal  upon  the  corpuscles  of  another  species  be  designated  as 
"  heterolysis,"  and  the  active  agent  or  agents  in  the  hemolytic 
serum  in  this  case  be  termed  "  heterolysins,"  while  the  hemolytic 
action  of  the  serum  of  one  species  on  the  corpuscles  of  another  indi- 
vidual of  the  same  species  be  designated  as  "  isolysis,"  and  the 
active  agents  as  "  isolysins."  If  a  serum  should  be  found  which 
dissolves  the  corpuscles  of  the  individual  from  which  the  serum  has 
been  obtained,  the  process  would  be  designated  "  autolysis,"  and  the 
active  agents  as  "  autolysins."  No  one  has  as  yet  discovered  an 
autolysin,  although  the  possibility  of  the  existence  of  such  a  sub- 
stance cannot  be  denied.  It  is  possible  that  certain  diseased  con- 
ditions, which  we  have  designated  as  auto-intoxications,  may  be 
caused  in  this  way. 

Ehrlich  designates  the  haptophorous  group  in  the  corpuscle,  or 
the  side-chain  in  the  corpuscle  which  combines  with  the  intermediary 
body,  as  the  "  receptor."  He  supposes  that  it  is  by  the  action  of  these 
receptors  that  hemolysins,  and  other  toxins  as  well,  combine  with 
and  destroy  the  cells  of  the  body.  He  illustrates  his  views  of  the 
relationship  between  the  cell,  the  intermediary  body  and  the  comple- 
ment, also  the  action  of  other  toxins,  by  the  accompanying  drawing. 
1  Berliner  Bin.  Woehemchrift,  1900,  453. 
9 


130 


THE  LYSINS. 


Fig.  1. 


-..JEJ 


DemonBtrating  Ehrlich's  Theory.    A,  complement ;  B,  intermediary  Body  ; 
part  of  cell ;  JS,  toxophorous  group  of  toxin  ;  F,  haptophorous  group. 


C,  receptor ;  B, 


If  there  be  no  receptors  the  intermediary  body  cannot  combine 
with  the  corpuscle  and  consequently  there  is  no  hemolysis.  Anti- 
hemolysins  are  supposed  to  be  formed  in  the  body  of  the  animal 
treated  with  hemolytic  serum  in  the  same  way  that  antitoxins  are 
formed  in  the  bodies  of  animals  immunized  to  the  toxins.  If  a 
hemolytic  serum  be  injected  into  an  animal  in  small  but  gradually 
increased  doses  at  intervals,  immunity  to  such  serum  is  obtained  and 
the  serum  of  the  animal  thus  immunized  contains  an  anti-hemolysin. 
When  a  small  amount  of  the  hemolytic  serum  is  injected  it  is  taken 
up  by  the  receptor  in  the  blood  cell,  and  provided  that  the  amount 
of  the  hemolytic  serum  injected  is  small,  the  blood  corpuscle  is  not 
destroyed ;  and  needing  for  the  performance  of  its  function  the  re- 
ceptor which  has  combined  with  the  hemolysin,  it  throws  out  other 
receptors ;  and  as  in  the  case  of  the  production  of  antitoxin,  a  point 
is  reached  when  there  is  over-production  of  receptors  and  those  not 
needed  by  the  cell  and  not  taken  up  by  the  hemolysin  are  cast  off  in 
the  blood  and  constitute  the  anti-hemolysin.  Ehrlich  and  Morgen- 
roth  prepared  an  anti-isolysin  in  the  following  manner  :  A  small 
goat  (No.  10)  whose  blood  corpuscles  had  been  shown  to  be  highly 
susceptible  to  the  serum  of  goat  A  was  treated  at  intervals  with  this 
serum  and  an  anti-body  obtained.  When  0.4  c.c.  of  the  serum  of  goat 
No.  10  was  added  to  one  c.c.  of  a  five  per  cent,  dilution  of  the  blood 
of  a  goat  which  had  been  found  susceptible  to  the  serum  of  goat  A, 
no  hemolysis  occurred.     However,  when  the  blood  corpuscles  of  goat 


THE  LTSINS.  131 

No.  10  were  freed  from  their  own  serum  and  washed  with  physiolog- 
ical salt  solution  they  were  found  to  be  as  susceptible  to  the  serum 
of  goat  A  as  they  were  before. 

Goat  B  was  treated  in  exactly  the  same  manner  as  goat  A.  How- 
ever, it  was  found  that  in  this  case  no  isolysin  appeared  in  the  serum 
until  the  fifteenth  day,  when  its  appearance  was  sudden.  During 
the  first  fourteen  days  after  the  injection  the  erythrocytes  of  goat  B 
remained  highly  susceptible  to  the  isolysin  in  the  serum  of  goat  A  ; 
and,  strange  to  say,  this  susceptibility  continued  even  after  the 
serum  of  goat  B  manifested  its  hemolytic  properties.  It  was  further- 
more found  that  the  erythrocytes  of  certain  goats  were  susceptible  to 
the  isolysin  of  goat  A  and  insusceptible  to  that  of  goat  B.  It  ap- 
pears from  this  that  there  are  different  isolysins  and  it  was  also  found 
that  anti-isolysin  A  was  wholly  without  action  against  isolysin  B. 

A  third  goat,  (7,  received  at  the  same  time  a  like  amount  of  the 
same  blood  as  B,  and  first  furnished  a  hemolysin  on  the  seventh  day. 
This  hemolysin  was  also  found  to  be  an  isolysin,  but  different  from 
that  of  either  A  or  B.  These  experiments  demonstrated  that  the 
exact  nature  of  the  isolysin  depends  upon  the  individual  character- 
istics of  the  animal  in  which  it  is  formed.  A  fourth  goat,  D,  fur- 
nished an  isolysin  which  dissolved  the  blood  corpuscles  of  B  and  C, 
but  was  without  effect  upon  those  of  A.  The  sera  oi  A,  B  and  C 
dissolved  sheep's  corpuscles,  while  the  serum  of  D  was  without 
effect. 

Ehrlich  compares  the  intermediary  body  with  diazo-benzaldehyde, 
which  by  means  of  its  diazo  group  is  capable  of  combining  with  a 
series  of  bodies,  such  as  aromatic  amins,  phenols,  keto-methyl  bodies, 
etc.,  while  by  means  of  its  aldehyde  group  it  may  combine  with  a 
different  series  such  as  the  hydrazins,  ammonia  radicles,  and  hydro- 
cyanic acid.  Phenol  and  hydrocyanic  acid  will  not  directly  combine, 
but  with  diazo-benzaldehyde  acting  as  an  intermediary  body,  these 
two  substances  can  be  brought  into  combination.  Pushing  this 
comparison  further,  we  may  say  that  the  aromatic  body,  or  the 
phenol,  represents  a  constituent  of  the  blood  corpuscle.  The  diazo- 
benzaldehyde  is  the  intermediary  body,  while  the  poisonous  hydro- 
cyanic acid  constitutes  the  complement.  As  has  been  stated,  the 
intermediary  body  has  two  haptophorous  groups.  By  means  of  one 
of  these  it  combines  with  the  receptor  of  the  cell,  while  by  means  of 
the  other  it  combines  with  the  complement.  The  former  may  be 
designated  as  the  cytophil  group,  while  the  other  may  be  distin- 
guished by  the  designation  of  complementophil.  It  should  be 
understood  that  there  are  probably  many  varieties  of  intermediary 
bodies,  and  there  may  be  two  or  more  in  a  given  blood  serum.  In 
fact,  Ehrlich  has  demonstrated  the  presence  of  two  or  more  kinds  of 
intermediary  bodies  in  the  same  serum.  The  serum  of  a  rabbit 
which  has  been  immunized  to  ox  blood  has  a  hemolytic  action  not 


132  THE  LYSINS. 

only  upon  the  corpuscles  of  the  ox,  but  upon  those  of  the  goat  as 
well.  If  such  a  blood  serum  be  treated  with  a  sufficient  amount  of 
the  corpuscles  of  the  ox,  and  the  mixture  separated  in  a  centrifuge, 
the  supernatant  fluid  has  no  solvent  action  upon  either  ox  or  goat 
corpuscles.  In  other  words,  the  ox  corpuscles  have  taken  up  all  the 
intermediary  bodies  in  the  blood  serum.  However,  if  this  serum  be 
mixed  with  goat  corpuscles  and  the  mixture  be  separated  in  a  cen- 
trifuge, the  supernatant  fluid  has  no  solvent  action  upon  goat 
corpuscles,  but  still  possesses  solvent  action  on  ox  corpuscles.  This 
experiment  demonstrates  that  there  are  in  the  blood  serum  of  a 
rabbit  immunized  to  ox  blood  at  least  two  intermediary  bodies,  both 
of  which  are  capable  of  combining  with  ox  corpuscles,  while  only 
one  combines  with  goat  corpuscles.  Intermediary  bodies  differ  from 
one  another  both  in  their  cytophil  and  in  their  complementophil 
groups.  A  given  intermediary  body  will  unite  with  a  corpuscle 
only  when  it  finds  in  that  corpuscle  its  appropriate  receptor.  And, 
as  has  already  been  shown,  the  receptors  in  one  and  the  same  cor- 
puscle, as,  for  instance,  in  the  corpuscle  of  the  ox,  differ  and  take  up 
different  intermediary  bodies.  In  like  manner,  every  intermediary 
body  will  not  combine  with  every  complement ;  and  combination 
between  an  intermediary  body  and  a  complement  can  occur  only 
when  their  haptophorous  groups  are  homologous.  The  existence  of 
two  or  more  kinds  of  intermediary  bodies  in  a  given  serum  has  been 
also  demonstrated  by  the  formation  of  anti-intermediary  bodies.  If 
a  hemolytic  serum  be  injected  in  small  quantities  at  intervals  into 
an  animal  there  may  be  obtained  from  that  animal  a  serum  which 
contains  an  anti-intermediary  body,  but  its  anti-action  is  not  mani- 
fest towards  all  intermediary  bodies  and  may  show  itself  only  when 
brought  into  contact  with  that  intermediary  body  by  means  of  which 
the  anti-hemolytic  serum  has  been  obtained,  and  it  is  in  this  sense 
only  that  specific  anti-intermediary  bodies  can  be  obtained.  A 
serum  containing  an  anti-intermediary  body  prevents  the  action  of  a 
hemolytic  serum  on  the  corpuscle  in  case  that  the  action  results  from 
its  own  specific  intermediary  body.  The  action  of  an  anti-inter- 
mediary body  consists  in  preventing  the  union  between  the  cytophil 
group  of  its  specific  intermediary  body  and  the  receptor  of  the  cell. 
It  is  possible  that  intermediary  bodies  possessing  different  cytophil 
groups  may  have  the  same  complementophil  group,  or  intermediary 
bodies  of  like  cytophil  groups  may  have  different  complementophil 
groups  ;  and  it  is  still  possible  that  there  may  be  intermediary 
bodies  possessed  of  only  one  cytophil  group  and  having  two,  three 
or  more  complementophil  groups.  In  his  latest  articles  Ehrlich 
designates  the  intermediary  body  as  "  amboceptor,"  indicating  that 
it  has  two  haptophorous  groups,  but,  as  has  just  been  stated,  the 
intermediary  body  may  be  a  triceptor,  quadriceptor,  etc. 

Each  complement  has  a  haptophorous  group  by  virtue  of  which  it 


THE  LYSINS.  133 

is  able  to  effect  a  combination  with  the  intermediary  body,  and  a 
zymotoxic  group  by  means  of  which  it  destroys  the  corpuscle.  Ehr- 
lich  has  shown  the  close  relationship  between  the  complement  among 
the  hemolytic  factors  and  the  bacterial  toxins,  inasmuch  as  he  has 
demonstrated  the  existence  of  complementoids  which  correspond  to 
the  toxoids.  As  has  already  been  stated,  many  of  the  complements 
are  deprived  of  their  poisonous  action  by  a  heat  of  56°.  Ehrlich 
has  shown  that  this  apparent  destruction  of  the  complement  by  heat 
consists  only  in  destroying  the  zymotoxic  group.  If  an  animal  be 
immunized  with  a  hemolytic  serum  which  has  been  rendered  inactive 
by  heat,  there  is  formed  in  this  animal  not  only  an  anti-intermediary 
body,  but  also  an  anti-complement.  This  procedure  is  analogous  to 
the  production  of  antitoxin  by  treating  an  animal  with  a  toxoid. 
The  anti-complement  produced  in  this  way  is  often  quite  as  potent 
as,  sometimes  more  so  than,  that  obtained  by  immunizing  an  animal 
with  an  unheated  or  active  hemolytic  serum,  just  as  a  highly  active 
antitoxin  may  be  obtained  by  treating  an  animal  with  a  relatively 
harmless  toxoid.  An  anti-complement  prevents  the  action  of  its 
specific  complement  by  rendering  it  impossible  for  the  latter  to  com- 
bine with  the  intermediary  body.  It  is  possible  that  complements, 
like  intermediary  bodies,  may  vary  in  the  number  of  haptophorous 
groups  which  they  possess.  If  an  intermediary  body  be  possessed 
of  two  complementophil  groups,  it  may  take  up  two  mono-haptoph- 
orous  complements,  or  one  di-haptophorous  complement. 

Bordet  ^  combats  Ehrlich's  view  that  the  union  between  the  inter- 
mediary body  and  the  blood  cell  is  a  chemical  one,  and  claims  that  it 
is  a  phenomenon  of  surface  absorption.  The  experiment  upon  which 
this  claim  is  based  may  be  detailed  as  follows  :  If  a  guinea-pig  be 
treated  with  rabbit's  blood,  there  is  produced  in  the  former  animal  a 
hemolytic  serum  which  will  dissolve  a  given  number  of  the  blood 
corpuscles  of  the  rabbit.  Having  determined  the  amount  of  blood 
which  a  given  quantity  of  this  serum  will  completely  dissolve,  pro- 
vided that  all  the  blood  is  added  at  one  time  to  the  serum,  Bordet 
added  to  the  determined  quantity  of  serum  half  this  amount  of  rab- 
bit's blood  and  then,  after  allowing  it  to  stand  for  a  while,  added 
the  other  half  of  the  blood,  when  it  was  found  that  the  serum  dis- 
solved only  that  portion  of  blood  which  was  first  added  and  had  no 
effect  upon  the  second  portion.  He  compares  this  to  the  following 
simple  experiment  in  surface  absorption  :  If  a  small  amount  of  methyl 
violet  be  dissolved  in  a  given  volume  of  water,  and  a  piece  of  filter 
paper  be  immersed  in  this  solution  for  a  short  time,  the  paper  will 
absorb  all  the  coloring  matter,  leaving  the  water  colorless.  Now,  if 
the  same  amount  of  methyl  violet  be  dissolved  in  the  same  amount 
of  water,  and  a  piece  of  filter  paper  half  the  size  of  that  used  in  the 
first  experiment  be  immersed  in  this  solution,  it  also  will  absorb  all 
^Annates  de  PInstitut  Pasteur,  1900. 


134  THE  LYSINS. 

the  coloriug  matter  and  leave  the  solution  colorless.  Of  course,  in 
the  latter  instance,  the  paper,  being  smaller,  will  be  more  highly- 
colored.  Ehrlich  has  repeated  Bordet's  experiment  with  hemolytic 
serum  and  blood  corpuscles  and  has  not  only  confirmed  his  results, 
but  has  shown  that  even  a  much  smaller  proportion  than  one-half 
the  corpuscles  will  combine  with  all  the  intermediary  body  present ; 
but  his  explanation  is  wholly  diflPerent  from  that  suggested  by  Bordet. 
Ehrlich  claims  that  when  half  the  corpuscles  are  added  to  the  amount 
of  serum  capable  of  digesting  the  whole  number  of  corpuscles,  each 
corpuscle  combines  with  a  larger  number  of  intermediary  bodies  than 
is  necessary  to  effect  solution,  but  the  combination  having  taken  place, 
the  intermediary  bodies  are  held  and  are  not  free  to  act  upon  the 
corpuscles  added  in  the  second  portion.  Ehrlich  states  that  reactions 
similar  to  this  are  well  known  in  chemistry  and  he  mentions  the  fol- 
lowing instance :  Naphthalin  consists  of  two  benzol  rings  linked 
together.  If  a  salt-forming  group,  a  hydroxyl  or  amido  group,  be 
brought  in  contact  with  naphthalin,  there  are  formed  hetero-nuclear 
substitution  products,  such  as  di-oxy-naphthalin,  amido-naphthol, 
and  naphthalin-diamin,  whose  sulpho-acids  are  capable  of  combining 
with  either  one  or  two  molecules  of  a  diazo-compound.  If  two 
molecules  of  dioxy-naphthalin  be  added  to  two  molecules  of  a  diazo- 
benzol,  there  is  formed  exclusively  a  mono-azo-compound ;  but  it 
two  molecules  of  diazo-benzol  be  added  to  one  molecule  of  dioxy- 
naphthalin,  there  is  formed  a  diazo-compound.  If  an  additional 
molecule  of  dioxy-naphthalin  be  added  to  the  already  formed  diazo- 
compound,  it  is  not  capable  of  decomposing  this  substance  ;  and  the 
diazo-compound  and  the  unchanged  dioxy-naphthalin  exist  together. 
We  have  already  referred  to  the  fact  that  Bordet  was  the  first  to 
show  that  the  addition  of  a  small  quantity  of  normal  serum  to  that 
of  an  animal  which  had  been  immunized  against  the  cholera  vibrio 
strengthened  the  bacteriolytic  action  of  the  cholera  serum.  This 
experiment  opened  up  the  way  by  which  it  has  been  ascertained  that 
there  are  two  factors  present  in  bacteriolytic  and  hemolytic  sera, 
inasmuch  as  the  normal  serum  used  by  Bordet  contained  the  sub- 
stance which  we  now  designate  "  the  complement,"  following  Ehr- 
lich's  theory.  In  a  second  series  of  experiments,  Bordet  ^  treated 
guinea-pigs  intravenously  with  five  or  six  successive  injections  of 
10  c.c.  each,  of  the  defibrinated  blood  of  the  rabbit.  From  the 
animals  thus  treated  he  obtained  a  serum  possessed  of  the  following 
characteristics  :  (1)  This  serum  when  mixed  with  the  defibrinated 
blood  of  the  rabbit  agglutinates  the  corpuscles  with  great  energy. 
For  example,  one  part  of  the  serum  agglutinated  all  the  red  blood 
corpuscles  contained  in  fifteen  parts  of  the  defibrinated  blood  of  the 
rabbit.  (2)  The  serum  not  only  agglutinated  but  subsequently 
rapidly  dissolved  the  blood  corpuscles  of  the  rabbit.     When  one 

*  Annales  de  I' InstUut  Pasteur,  1898. 


THE  LTSINS.  136 

part  of  the  defibrinated  blood  of  the  rabbit  was  added  to  two  or 
three  parts  of  the  active  serum,  the  mixture  became,  within  two  or 
three  minutes,  red,  clear  and  limpid.  Microscopical  examination  of 
this  fluid  showed  only  the  stroma  of  the  globules,  more  or  less  de- 
formed, transparent,  and  scarcely  visible.  (3)  When  the  active 
serum  of  the  guinea-pig  was  heated  to  55°  for  half  an  hour  (or  for 
less  time  at  60°)  it  lost  its  property  of  dissolving  the  corpuscles  of 
the  rabbit  but  still  agglutinated  them.  (4)  If  to  a  mixture  of  the 
defibrinated  blood  of  the  rabbit  and  the  serum  of  the  guinea-pig, 
the  latter  having  been  heated  to  55°,  there  was  added  a  certain 
quantity  of  fresh  serum  from  either  a  normal  guinea-pig  or  rabbit 
the  hemolytic  property  of  the  serum  was  restored.  (5)  It  follows 
from  the  above  that  the  destruction  of  the  hemolytic  properties  of 
the  serum  of  the  guinea-pig  is  only  partial  and  the  injury  done  this 
serum  by  heat  is  partially  at  least  repaired  by  the  addition  of  fresh 
serum  from  either  a  normal  rabbit  or  a  normal  guinea-pig.  (6)  The 
serum  of  an  untreated  guinea-pig  has  only  feeble  agglutinating 
power  on  the  corpuscles  of  the  rat  and  has  no  solvent  action  on 
these  bodies.  (7)  The  active  serum  of  the  treated  guinea-pig  was 
without  influence  on  the  defibrinated  blood  of  another  guinea-pig, 
also  without  action  on  the  corpuscles  of  the  pigeon.  It  agglu- 
tinated markedly  the  corpuscles  of  the  rat  and  the  mouse.  (8) 
When  two  c.c.  of  the  defibrinated  blood  of  the  rabbit  were  intro- 
duced into  the  peritoneal  cavity  of  a  guinea-pig  which  had  been 
immunized  to  th  e  blood  of  the  rabbit,  the  corpuscles  thus  introduced 
were  quickly  and  completely  destroyed.  The  liquid  removed  from 
the  cavity  after  about  ten  minutes  was  found  to  be  uniformly  red 
and  perfectly  clear  and  limpid.  (9)  If  the  blood  of  the  rabbit 
mixed  with  a  small  quantity  of  the  serum  of  the  treated  guinea- 
pig  which  had  been  heated  to  55°  was  injected  into  the  peritoneal 
cavity  of  the  treated  guinea-pig,  the  phenomenon  of  destruction 
took  place  very  rapidly.  (10)  The  serum  of  the  guinea-pig  injected 
intravenously  into  the  rabbit  proved  to  be  highly  toxic. 

Bordet  ^  has  made  additional  valuable  contributions  to  the  subject 
of  hemolysis.  The  facts  ascertained  in  these  researches  agree  prac- 
tically in  all  details  with  those  already  given  concerning  the  work  of 
Ehrlich  and  Morgenroth,  but  Bordet's  explanation  of  the  facts 
difi'ers  from  that  given  by  the  German  investigators.  Ehrlich's 
intermediary  body  is  designated  by  Bordet  as  the  sensitizer  (substance 
sensibilatHce),  and  its  function  is  to  render  impressionable  globules 
sensitive  to  the  action  of  the  toxic  body,  which  Ehrlich  designates  as 
complement  and  Bordet  calls  alexin.  Ehrlich  holds  that  the  combi- 
nation between  the  corpuscles  and  the  intermediary  body  is  a  chem- 
ical one,  while  Bordet  explains  the  action  of  his  sensitizer  on  phys- 
ical grounds.  The  term  alexin,  adopted  by  Bordet,  is  the  same  as 
^Annales  de  I'lmtitut  Pasteur,  1899  and  1900. 


136  THE  LYSINS. 

that  used  by  Buchner  to  indicate  the  germicidal  constituent  of  blood 
serum.  Bordet  has  also  prepared  anti-sensitizers  and  anti-alexins 
by  immunizing  animals  to  hemolytic  sera.  It  will  be  seen  from  this 
that  there  is  but  little  difference  either  in  the  experimental  results 
obtained  or  in  the  theoretical  explanation  offered  by  the  German  and 
the  French  investigators.  It  should  also  be  mentioned  that  it  is 
generally  believed  that  the  combination  between  the  intermediary 
body  and  the  blood  corpuscle,  whether  it  be  chemical  or  physical,  is 
confined,  so  far  as  the  corpuscle  is  concerned,  to  the  stromata,  and 
that  the  hemoglobin  takes  no  part  in  the  reaction.  Miiller  calls  the 
intermediary  body  "  copula,"  London  designates  it  "  desmon,"  while 
Metschnikoff  calls  the  intermediary  body  "  philocytase "  and  the 
complement  "  cytase." 

It  has  long  been  known  that  the  sera  of  certain  animals  may  dis- 
solve the  red  blood  corpuscles  of  animals  of  other  species.  So  far 
as  we  know,  the  first  recorded  observation  of  this  phenomenon  was 
made  by  Dumas  and  Prevost.^  Early  experiments  on  the  transfusion 
of  blood  from  one  animal  to  another  gave  opportunity  for  repeated 
observation  of  hemolytic  effects.  One  of  the  first  attempts  to  study 
hemolysis  by  the  methods  of  exact  scientific  examination  was  made 
by  Ehrlich  ^  in  1884,  when  he  disproved  the  generally  held  idea  that 
cold  is  a  causative  factor  in  the  production  of  hemoglobinuria.  He 
closed  his  paper  on  this  subject  by  suggesting  that  cold  can  lead  to 
the  dissolution  of  blood  corpuscles  only  in  specially  disposed  indi- 
viduals in  which  the  walls  of  the  blood  vessels  produce  certain  agents 
(ferments?)  which  injure  the  '' discoplasma."  In  1898  Belfanti 
and  Carbone  ^  ascertained  that  the  blood  serum  of  an  animal  treated 
with  the  blood  of  an  animal  of  another  species  proved  toxic  when 
injected  intravenously  into  the  animal  from  which  the  blood  had 
been  originally  obtained.  Rabbit's  blood  was  injected  subcutaneously 
into  horses  and  the  serum  of  the  horse  injected  into  the  rabbit 
caused  dissolution  of  the  corpuscles  and  induced  death.  This  ob- 
servation was  probably  the  starting  point  of  the  numerous  experi- 
mental studies  which  have  been  made  upon  this  subject  within  the 
last  two  years.  Almost  simultaneously  Bordet  in  France  and  Land- 
steiner  in  Austria  published  the  results  of  experiments  along  this 
line.  We  have  already  referred  to  Bordet's  work,  and  that  of  Land- 
steiner  *  contributed  nothing  specially  new.  Von  Duugern  ^  injected 
the  blood  of  chickens  and  pigeons  into  the  peritoneal  cavities  of 
guinea-pigs  and  observed  that  at  first  the  corpuscles  thus  injected 
were  slowly  dissolved,  but  upon  repeating  the  injections  into  the 
same  animals  he  found  that  solution  occurred  more  promptly  and 

*  Anncdes  de  Chimie,  1821. 

2  Charite-Annakn,  10. 

3  Oiornal  d.  R.  Acad,  di  Med.  di  Torino,  1898. 

*  Centralblall  f.  Bakteriologie,  25. 

5  Miinchener  med.  Wochenschrift,  1899. 


PLATE  I. 


Ehruch's  Figures — Illustrating  his  Theory. 


I- -A 


Fig.  1. 
a.   Complement ;    b.    Interme- 
diary body;  c.  Receptor;  d.  Part 
of  cell. 


0 


Fig.  2. 
Cell  with  different  kinds  of  receptors,     c.    Re- 
ceptors. 


Fig.  3. 
Showing  separation   of  antitoxins,    and 
combination  of  toxins  with  free  antitoxins. 


Fig.  4. 
Showing  the  action  of  anti-comple- 
ment,     a.    Complement ;     b.    Inter- 
mediary body  ;  c.  Receptor  ;  d.  Cell. 
e.  Anti-complement. 


THE  LYSINS.  137 

that  the  serum  of  the  guinea-pig  agglutinated  and  dissolved  the  cor- 
puscles of  the  birds  both  inside  and  outside  the  body.  Metschni- 
koff  ^  repeated  these  experiments  using  the  blood  of  the  goose,  and 
Krorapecber  ^  has  done  the  same  with  the  corpuscles  of  the  frog.  It 
should  be  stated  that  there  is  some  advantage  in  the  use  of  nucleated 
red  cells  for  injection  purposes,  inasmuch  as  the  nuclei  are  not  dis- 
solved and  the  number  of  free  nuclei  is  an  index  to  the  degree  ot 
hemolysis.  If  to  a  drop  of  the  serum  of  a  rabbit  which  has  been 
immunized  to  chicken's  blood,  there  be  added  a  small  quantity  of 
chicken  blood  and  guinea-pig  blood,  and  the  mixture  be  examined 
under  the  microscope,  it  will  be  seen  that  at  first  the  nucleated  cells 
of  the  chicken  blood  agglutinate  while  the  cells  of  the  guinea-pig 
blood  remain  evenly  distributed  and  later  the  chicken  blood  under- 
goes hemolysis,  while  that  of  the  guinea-pig  remains  unaffected. 

Mosso  ^  found  that  the  popular  belief  that  the  blood  of  the  eel  is 
poisonous  to  mammals  is  true,  and  he  demonstrated  that  the  intrave- 
nous injection  of  0.1  c.c.  of  eel  serum  per  kilogram  of  body  weight  into 
rabbits  and  guinea-pigs  induced  death  within  three  minutes  and  that 
post-mortem  examination  showed  that  the  mammalian  erythrocytes  had 
been  dissolved.  Subsequent  investigation  showed  that  the  hemolytic 
action  of  eel  serum  on  the  blood  of  mammals  may  be  demonstrated 
in  vitro.  Kossel,*  Camus  and  Gley,^  and  Tchistovitch  ^  took  up 
this  investigation  and  showed  that  rabbits,  dogs  and  goats  could  be 
easily  immunized  against  eel  serum  and  that  the  antitoxin  thus  ob- 
tained prevented  the  hemolytic  action  of  this  serum  in  vitro,  as  well 
as  in  vivo. 

Camus  and  Gley  ^  have  shown  that  the  blood  corpuscles  of  some 
animals  (hedgehogs  and  certain  birds)  are  not  susceptible  to  the 
hemolytic  action  of  eel  serum  and  they  explain  this  by  stating  that 
it  is  due  to  a  peculiarity  of  the  organization  of  the  blood  cells  of  these 
animals.  According  to  Ehrlich's  theory,  it  might  be  said  that  the 
red  blood  corpuscles  of  these  animals  have  no  receptors  with  which 
the  toxic  substance  in  eel  serum  can  combine.  The  venoms  of  the 
cobra  and  other  poisonous  snakes  have  a  marked  hemolytic  effect 
upon  the  blood  of  mammals  which  can  be  arrested  by  the  action  of 
specifically  prepared  anti-hemolytic  sera. 

Halban*  has  made  an  important  contribution  to  our  knowledge  of 
both  agglutinins  and  hemolysins,  and  although  the  agglutinins  will  be 
discussed  in  a  subsequent  chapter,  in  order  to  save  repetition  we  will 

'  Annales  de  V  Institut  Pasteur,  1900. 
^  Centralblattf.  Bakteriologie,  28. 
^Archivf.  Experiment.  Pathologie,  25. 

*  Berliner  klin.  Wochenschrift,  1898. 

*  Archiv  Intemat.  de.  Pharmacodynamie,  3  and  4. 
^Annales  de  F Institut  Pasteur,  1899. 

'' Compt.  Rend,  del' Acad.  deSc,  1899. 

*  Wiener  klin.  Wochenschrift,  1900. 


138  I  HE  LYSINS. 

give  a  condensed  statement  here  concerning  Halban's  work  with  both 
of  these  substances.  He  obtained  sterile  fetal  blood  drawn  from  the 
placental  end  of  the  cord  cut  directly  after  birth,  while  the  mother's 
blood  was  secured  by  placing  small  sterile  dishes  in  the  vagina  after 
the  removal  of  the  placenta.  He  prepared  from  each  kind  of  blood 
both  defibrinated  blood  and  sera.  With  these  preparations  he  tested 
the  agglutinating  and  hemolytic  effects  of  the  serum  of  the  fetus  on  its 
own  mother,  and  vice  versa;  also  the  action  of  the  fetal  serum  on  the 
blood  of  other  mothers.  The  results  of  these  investigations  may  be 
summed  up  as  follows  :  (1)  The  agglutinating  properties  of  the  blood 
of  the  fetus  did  not  appear  to  be  dependent  upon  those  of  the  mother's 
serum,  inasmuch  as  the  mother's  serum  frequently  agglutinated  mark- 
edly, while  the  serum  of  the  fetus  did  not  agglutinate  at  all.  Occa- 
sionally this  condition  was  reversed,  but  as  a  rule  the  agglutinating  ac- 
tion of  the  fetal  blood  was  much  less  marked  than  that  of  the  maternal. 
(2)  A  like  result  was  obtained  when  the  action  of  both  kinds  of  serum 
was  tested  upon  the  blood  of  the  mothers  of  other  infants.  (3)  The 
same  differences  were  observed  as  a  rule  when  the  sera  were  mixed 
with  the  blood  of  other  individuals.  (4)  In  no  case  did  the  serum 
of  either  fetus  or  mother  agglutinate  the  corpuscles  of  the  individual 
from  which  the  serum  was  obtained.  (5)  In  a  considerable  number 
of  cases  the  serum  of  the  mother  agglutinated  the  blood  of  the  fetus 
and  the  serum  of  the  fetus  agglutinated  the  blood  of  the  mother.  In 
other  words,  the  two  sera  acted  in  this  respect  as  did  sera  obtained 
from  different  individuals.  When  the  serum  of  the  mother  aggluti- 
nated the  blood  of  other  individuals  it  also  agglutinated  that  of  her 
own  child ;  and,  likewise,  when  the  serum  of  the  fetus  agglutinated 
the  blood  of  other  individuals  it  also  agglutinated  the  blood  of  its 
own  mother.  (6)  The  hemolytic  action  of  both  sera  as  a  rule  par- 
alleled their  agglutinating  effects.  (7)  The  agglutinating  action  of 
both  sera  was  tested  on  cholera  cultures  and  showed  the  same  reac- 
tion with  these  cells  as  with  red  blood  corpuscles.  (8)  These  inves- 
tigations show  that  the  kind  and  amount  of  agglutinins  and  lysins 
existing  in  the  blood  of  the  mother  and  the  fetus  differ  as  they  would 
between  other  individuals.  From  these  studies  Halban  draws  con- 
clusions which  may  be  condensed  as  follows  : 

A.  The  fact  that  the  maternal  and  fetal  blood  belong  to  two 
wholly  distinct  circulatory  systems  is  established.  It  is  known  that 
the  chemical  properties  of  these  two  bloods  differ,  and  Kriiger  has 
shown  that  the  fetal  blood  contains  only  slightly  more  solid  con- 
stituents than  the  maternal  blood  ;  while  the  fibrin  of  the  former  is 
markedly  less  than  that  of  the  latter.  Scherenziss  has  demonstrated 
that  the  specific  gravity  of  fetal  blood  is  somewhat,  and  that  of 
fetal  serum  markedly,  lower  than  that  of  the  mother.  Fetal  blood 
contains  less  hemoglobin  and  therefore  its  red  corpuscles  must  be 
richer  in  stroma  and  are  more  easily  destroyed  than  the  corpuscles 


THE  LYSINS.  139 

of  the  mother.  On  the  other  hand,  fetal  blood  is  richer  in  inorganic 
salts,  especially  the  insoluble  salts,  than  is  the  blood  of  adults. 
Furthermore,  fetal  blood  is  richer  in  sodium,  poorer  in  potassium, 
and  the  total  quantity  of  chlorin  not  combined  with  either  potassium 
or  sodium  is  markedly  smaller  than  in  the  blood  of  adults. 

B.  It  also  follows  from  these  experiments  that  the  maternal  blood 
often  contains  agglutinins  while  the  fetal  blood  shows  no  trace  of 
these  substances  ;  and  inasmuch  as  the  fetus  must  obtain  albuminous 
substances  from  the  maternal  blood  in  the  construction  of  its  own 
tissue,  and  as  the  agglutinins  are  supposed  to  belong  to  the  globulins, 
it  follows  that  all  albuminous  substances  are  not  absorbed  by  the 
fetus  from  the  mother  equally  and  that  there  must  be  a  selective 
absorption  manifested  by  the  epithelium  of  the  chorion. 

C.  This  investigation  bears  upon  the  question  of  the  origin  ot 
normal  agglutinins  in  the  blood ;  of  course,  it  has  nothing  to  do 
with  specific  agglutinins  produced  by  inoculation.  Since  agglutinins 
are  present  in  the  blood  of  the  mother,  it  has  been  supposed  that 
those  found  in  the  blood  of  the  fetus  have  simply  been  transferred 
from  the  former  to  the  latter,  but  the  above-mentioned  experiments 
contradict  this  view,  inasmuch  as  it  was  shown  in  some  instances 
that  there  are  agglutinins  in  fetal  blood  while  they  are  not  present 
in  the  blood  of  the  mother.  We  must,  therefore,  suppose  that  these 
substances  originate  independently  of  the  mother,  and  we  should 
distinguish  between  inborn  and  hereditary  agglutinins  and  lysins. 
Ehrlich  and  Morgenroth  have  proved  the  existence  of  isoagglutinins 
and  isolysins,  but  it  is  claimed  by  Halban  that  these  must  be  sub- 
divided into  ordinary  isoagglutinins  and  isolysins  on  one  hand,  and 
idio-isoagglutinins  and  idio-isolysins  on  the  other,  meaning  by  the 
last  given  terms  his  inborn,  non-hereditary  substances.  To  account 
for  the  origin  of  these  idio-iso-substances,  Halban  offers  the  follow- 
ing theories  :  (1)  The  idio-iso-agglutinins  and  idio-isolysins  are  due 
to  an  interchangeable  immunization  between  mother  and  fetus. 
Blood  cells  in  both  circulatory  systems  are  constantly  disintegrating 
and  the  products  of  their  disintegration  may  pass  from  mother  into 
fetus  and  vice  versa,  and  by  this  means  an  interchangeable  immuni- 
zation may  be  produced.  The  fact  that  the  agglutinins  are  more 
frequent  in  the  blood  of  the  mother  than  in  that  of  the  fetus  may 
be  explained  on  the  supposition  that  the  fetus  does  not  form  agglu- 
tinins so  readily.  Halban  supposes  that  this  theory  has  some  sup- 
port in  the  demonstrated  fact  that  children  infected  with  typhoid 
fever  during  the  first  year  of  life  as  a  rule  show  the  Widal  reaction 
less  promptly  than  adults  do ;  and  he  states  that  it  has  been  shown 
by  Schreiber  that  newly  born  infants  bear  without  apparent  effect 
doses  of  tuberculin  which  induce  in  adults  marked  elevation  of  tem- 
perature. Halban  admits  that  the  presence  of  agglutinins  in  the 
blood  of  women  who  have  never  borne  children,  and  also  in  the 


140  THE  LYSINS. 

blood  of  men,  is  difficult  of  explanation  by  this  theory,  unless  we 
assume  that  every  individual  is  possessed  of  the  agglutinins  and 
lysins  which  originated  in  that  individual  during  fetal  life  by  inter- 
changeable immunization  with  the  mother.  This  explanation  pro- 
vides for  an  existence  of  these  substances  more  prolonged  than  seems 
possible.  (2)  The  origin  and  continued  existence  of  agglutinins  and 
lysins  in  the  body  may  be  explained  on  the  assumption  that  they  are 
due  to  the  frequent  absorption  of  bacterial  substances,  especially  from 
the  intestines ;  but  the  fetus  has  been  found  to  be  germ-free,  and  it 
has  been  shown  by  Kraus  and  Clairmont  that  the  serum  of  newly- 
hatched  pigeons  often  possesses  very  marked  bacteriolytic  action. 
(3)  There  remains  the  possibility  that  nrormal  agglutinins  and  lysins 
may  be  due  to  self-immunization,  resulting  from  the  continued  dis- 
solution of  cells  in  the  body.  It  is  true  that  Ehrlich  and  Morgen- 
roth  did  not  succeed  in  producing  an  autolysin.  Halban  states  that 
there  are  present  in  normal  blood  not  only  idio-isoagglutinins  and 
idio-isolysins,  but  also  idio-heteroagglutinins  and  idio-heterolysins. 
The  presence  of  the  last-mentioned  substances  is  indicated  by  the 
property  of  normal  blood  of  agglutinating  and  dissolving  certain 
foreign  cells,  also  the  blood  corpuscles  of  other  species  of  animals. 
These  substances  can  hardly  be  supposed  to  have  their  origin  in  self- 
immunization.  So  long  as  we  are  unable  by  experimental  means  to 
produce  auto -agglutinins  and  auto-isolysins,  it  must  be  assumed  that 
the  agglutinins  and  lysins  of  normal  serum  are  inborn  substances. 

Meltzer,^  having  ascertained  that  the  normal  serum  of  the  ox  has 
a  marked  hemolytic  action  on  the  erythrocytes  of  the  rabbit,  placed 
this  serum  in  the  peritoneal  cavities  of  rabbits,  and,  removing  it  after 
varied  intervals,  compared  its  hemolytic  action  on  the  corpuscles  of 
the  rabbit  with  that  manifested  by  the  same  serum  before  it  was  in- 
troduced into  the  animal.  These  experiments  led  to  the  following 
conclusion  :  "  The  normal  hemolytic  power  of  bullock's  serum  for 
the  red  blood  corpuscles  of  the  rabbit  disappears  during  a  stay  in  the 
peritoneal  cavity  of  this  animal ;  and  the  disappearance  is  the  greater 
the  longer  the  stay,  and  is  independent  of  the  absorption  of  the  fluid  ; 
disappearance  takes  place  even  during  the  first  fifteen  minutes." 
Further  investigation  showed  that  the  disappearance  of  the  hemo- 
lysin was  not  due  to  the  formation  of  an  anti-hemolysin,  and  that  the 
hemolytic  action  of  the  ox  serum  was  lost,  but  more  slowly,  when  it 
was  introduced  into  the  peritoneal  cavity  of  a  dead  rabbit.  From 
this,  Meltzer  concluded  that  the  disappearance  of  the  hemolytic 
power  of  the  serum  was  due  to  the  imbibition  of  one  of  the  hemolytic 
factors,  and  subsequent  investigation  showed  that  the  factor  absorbed 
is  the  complement ;  however,  he  was  not  able  to  regenerate  the 
serum  removed  from  the  cavity  by  the  addition  of  sera  containing 
various  complements.  Meltzer  also  ascertained  that  hemolytic  serum 
» Medical  Record,  60,  1901. 


THE  LYSINS.  Ul 

obtained  by  immunization  becomes  inactive  when  kept  for  a  few 
hours  in  the  peritoneal  cavity ;  but  in  this  case,  regeneration  was 
accomplished  by  the  addition  of  fresh  serum  containing  a  comple- 
ment. In  these  experiments  rabbits  were  treated  intravenously  with 
guinea-pig's  blood  and  after  a  serum  had  been  obtained  which  had  a 
marked  hemolytic  action  on  the  blood  corpuscles  of  the  guinea-pig 
some  of  it  was  introduced  into  the  peritoneal  cavity  of  a  normal 
rabbit,  left  there  for  three  hours,  and  then  recovered.  "  Such  peri- 
toneal serum,  when  added  to  guinea-pig's  blood,  agglutinated  it,  but 
caused  no  hemolysis  whatsoever.  Addition  of  immunized  rabbit 
serum,  made  inactive  by  heating,  to  peritoneal  serum,  had  no  regen- 
erating effect.  But  addition  of  fresh  normal  rabbit  serum  to  the  im- 
munized peritoneal  serum  regenerated  it  completely  ;  the  mixture  of 
both  sera  dissolved  guinea-pig's  blood  as  readily  as  the  active  im- 
munized serum  alone." 

In  his  studies  of  cobra  lysin,  Myers  ^  found  that  when  this  toxin 
is  treated  with  anti-venomous  serum  it  behaves  similarly  to  the  bac- 
terial toxins.  For  instance,  it  was  ascertained  that,  in  order  to  neu- 
tralize 1  mg.  of  cobra  poison  so  that  it  would  no  longer  manifest  a 
hemolytic  action  on  the  blood  corpuscles  of  man,  1.3  c.c.  of  anti- 
venomous  serum  was  required ;  but  when  0. 1  c.c.  of  the  serum  was 
added  to  1  mg.  of  the  venom,  it  deprived  it  of  four-fifths  of  its  toxic 
action,  and  when  0.2  c.c.  of  the  serum  was  used  it  destroyed  nine- 
tenths  of  the  hemolytic  power  of  the  venom.  If  these  figures  be 
compared  with  those  given  by  Madsen  in  his  studies  of  tetanolysin 
and  already  referred  to  on  page  64,  the  similarity  will  be  evident. 
Myers  found  that  the  hemolytic  action  of  a  sample  of  dry  venom  re- 
mains constant  for  a  long  time,  but  such  a  venom  in  dilute  solution 
rapidly  decreases  in  hemolytic  action  until  it  reaches  a  minimum, 
when  there  is  no  further  change.  This  is  evidence  that  bodies  simi- 
lar to  the  toxoids  described  by  Ehrlich  are  also  formed  in  cobra 
lysin. 

Bulloch  and  Hunter^  have  shown  that  filtered  cultures  of  the  ba- 
cillus pyocyaneus  dissolve  the  red  corpuscles  of  the  ox,  the  sheep, 
rabbit,  monkey,  cat,  dog  and  rat.  The  more  concentrated  the  solu- 
tion of  the  toxin  the  more  rapidly  does  its  hemolytic  action  manifest 
itself,  but  in  all  the  experiments  there  appeared  to  be  a  latent  period 
during  which  there  was  no  hemolysis.  A  goat  was  immunized  with 
filtrates  of  this  bacillus,  and  in  the  serum  of  this  animal  there  ap- 
peared an  anti-hemolytic  substance  which  manifested  its  neutralizing 
effects  in  vitro.  In  experimenting  with  this  substance  it  was  ob- 
served that  while  small  quantities  of  the  immune  serum  did  not  pre- 
vent hemolysis  and  medium  quantities  did,  excessively  large  amounts 
of  the  serum  led  to  renewed  hemolytic  action. 

'  IVansactions  of  the  Pathological  Society  of  London,  51. 
^  Ibidem. 


142  THE  LYSINS. 

Von  Dungern  ^  discovered  that  epitheliolysins  may  be  produced. 
His  method  of  experimentation  and  the  results  obtained  may  be 
briefly  stated  as  follows :  He  removed  the  trachea  from  an  ox  imme- 
diately after  death,  and  from  this  he  scraped  the  ciliated  epithelial 
cells,  taking  care  to  avoid  mixture  with  red  blood  corpuscles  or 
connective  tissue.  The  epithelial  cells  thus  obtained  were  suspended 
in  physiological  salt  solution  and  injected  into  the  peritoneal  cavities 
of  guinea-pigs.  From  time  to  time  some  of  the  injected  cells  were 
removed  and  examined  microscopically.  The  first  change  observed 
was  one  of  form,  in  which  the  cells  were  found  to  be  rolled  up  like 
balls,  but  were  still  capable  of  moving  through  the  fluid  by  the  ac- 
tivity of  their  cilia.  Next  the  epithelial  cells  were  found  to  be 
clumped  in  masses,  with  the  cilia  still  active  and  many  of  these 
masses  were  seen  moving  like  an  individual  through  the  fluid. 
Later  the  cells  were  seen  to  undergo  a  cystoid  degeneration.  One 
or  more  small  vacuoles  could  be  seen  in  the  protoplasm,  and  these 
often  contained  leucocytes.  These  cysts  gradually  became  larger, 
pressing  the  nuclei  to  the  edges,  and  the  cells  appeared  like  large 
vacuoles  surrounded  by  thin  walls.  From  the  cavity  of  an  animal 
treated  in  this  way  for  the  first  time  visible  cells  can  be  obtained  in 
some  cases  as  long  as  six  or  even  ten  days  after  the  injection,  but 
when  the  same  animal  is  treated  after  a  lapse  of  ten  or  twelve  days 
with  a  second  injection  of  ciliated  epithelium  the  cells  are  found  to 
bedestroyed  with  much  greater  rapidity,  and  after  eighteen  hours  no 
eel  Is  with  motion  can  be  discovered.  By  continuing  the  immuniza- 
tion an  increased  solvent  action  on  the  part  of  the  peritoneal  fluid 
was  observed.  From  animals  thus  immunized  to  ciliated  epithelium 
there  was  obtained  a  serum  which  in  vitro  dissolved  similar  cells. 
Epithelial-immune  serum  dissolves  the  red  blood  corpuscles  as  well  as 
the  tracheal  epithelium  of  the  ox.  On  the  other  hand,  however,  the 
serum  of  guinea-pigs  immunized  to  ox  blood,  has  no  solvent  action 
upon  the  tracheal  epithelium  of  the  ox,  either  in  vitro  or  in  vivo. 
This  phenomenon  can  be  explained  in  two  ways.  Either  the  epithe- 
lial immune  serum  contains  two  lysins,  one  of  which  acts  upon  epi- 
thelial cells,  while  the  other  exerts  its  eflFect  wholly  on  the  erythro- 
cytes ;  or  there  may  be  in  the  epithelial  immune  serum  only  one 
lysin,  which  has  a  specific  action  on  ciliated  epithelium  and  a  non- 
specific action  on  the  erythrocytes.  That  the  latter  is  the  correct 
explanation  is  shown  by  the  fact  that  when  immune  serum  was 
brought  in  contact  with  a  mixture  of  epithelial  cells  and  blood  cor- 
puscles, it  acted  only  on  the  epithelium.  Evidently  the  lysin  in 
epithelial-immune  serum  has  a  greater  affinity  for  epithelial  cells  than 
it  has  for  blood  corpuscles,  and  when  the  former  of  these  is  present 
in  sufficient  quantity  in  a  mixture  of  the  two,  all  of  the  lysin  is  used 
up  in  effecting  a  combination  with  the  epithelial  cells.  Von  Dun- 
1  Miinchener  med.  Wochenschrift,  1899. 


THE  LYSINS.  143 

gern  suggests  the  possibility  of  using  an  epitheliolysin  in  the  de- 
struction of  the  unrecognizable  cancer  cells  that  may  remain  in  the 
tissue  after  excision  by  the  surgeon. 

In  a  second  communication  von  Dungern^  has  shown  that  rabbits 
and  guinea-pigs  immunized  to  cow's  milk  produce  sera  which  act 
upon  ciliated  epithelial  cells  and  also  possess  slight  hemolytic 
properties. 

Lindemann,^  in  carrying  out  his  investigations  concerning  the 
action  of  vinylamin  on  the  kidneys  recalled  the  fact,  discovered  by 
Claude  Bernard,  that  the  serum  of  an  animal  injected  intravenously 
into  an  animal  of  another  species  induces  a  more  or  less  permanent 
albuminuria.  This  led  him  to  endeavor  to  strengthen  the  action  of 
a  heterologous  serum  by  treating  the  animal  with  an  emulsion  of  the 
kidney  substance.  The  kidney  of  a  rabbit  was  rubbed  up  into  an 
emulsion  and  this  was  introduced  into  the  peritoneal  cavity  of  a 
guinea-pig.  From  the  latter  animal  there  was  obtained  a  serum 
which  when  injected  intravenously  into  rabbits  caused  rapid  destruc- 
tion of  the  kidneys  of  these  animals.  The  following  experiment 
illustrates  the  work  done  :  A  rabbit  which  had  received  without 
apparent  effect  eight  c.c.  of  the  serum  of  a  normal  guinea-pig,  was 
seven  days  later  given  a  like  amount  of  the  serum  of  a  guinea-pig 
which  had  been  treated  with  the  renal  emulsion.  The  latter  injec- 
tion provoked  albuminuria  within  a  few  hours,  followed  within  two 
days  by  complete  anuria  and  death.  The  kidneys  of  this  rabbit 
presented  histological  lesions  closely  resembling  those  found  in  ani- 
mals killed  by  the  administration  of  renal  poisons.  There  was 
marked  disintegration  and  necrosis  of  the  epithelium  of  the  convo- 
luted tubules.  The  greater  part  of  the  tubules  were  transformed 
into  granular  masses  containing  pycnotic  nuclei.  The  nuclei  which 
persisted  were  not  found  to  show  evidences  of  chromatolysis,  which 
is  characteristic  of  coagulation  necrosis,  induced  by  metallic  salts, 
but  they  resemble  those  found  after  poisoning  with  vinylamin  and 
in  which  the  alterations  in  the  protoplasm  precede  changes  in  the 
nuclei.  The  glomeruli  did  not  present  any  specific  alteration. 
Numerous  granular  casts  were  found  in  the  straight  tubules.  The 
serum  of  animals  treated  with  emulsions  of  kidney  is  said  to  be 
nephrolytic,  and  the  specific  toxin  contained  in  such  serum  is  known 
as  nephrolysin. 

In  a  second  communication  Lindemann  ^  found  that  the  serum  of 
dogs  which  were  suffering  from  nephritis  induced  by  intravenous  in- 
jections of  chromate  of  potash,  has  marked  nephrolytic  effect  when 
injected  into  other  dogs  and  causes  a  severe  nephritis  terminating  in 
death.     Schiitze,*  by  treating  rabbits  with  emulsions  of  the  kidneys 

'  Miinchener  med.   Wochenschrift,  1 900. 
^Annates  de  V Institut  Pasteur,  14,  1900. 
3  Ceniralblattf.  Pathologic,  1900. 
*  Deutsche  med.  Wochenschrift,  1900. 


144  THE  LYSINS. 

and  liver  of  normal  guinea-pigs,  was  not  able  to  secure  a  kidney-  or 
liver-serum  which  had  a  specific  effect  upon  homologous  cells. 
Nefediefi  has  gone  over  this  work  with  great  care  and  has  not  only- 
confirmed  all  the  statements  made  by  Lindemann,  but  has  pushed 
the  investigation  further  along  interesting  lines.  He  tied  the  ureter 
of  one  side  in  each  of  two  healthy  rabbits.  These  animals  bore  the 
operation  well,  the  wounds  healed  without  suppuration,  and  there 
was  marked  gain  in  weight.  Twenty-four  days  after  the  ligature 
had  been  applied  he  took  blood  from  the  artery  of  one  of  these 
animals  and  injected  the  serum  obtained  from  it  (four  c.c.  per  kilo) 
into  the  auricular  vein  of  a  healthy  rabbit.  The  urine  of  the  rab- 
bit thus  treated  contained  on  the  next  day  a  notable  quantity  of 
albumin  which,  however,  progressively  diminished  and  disappeared 
after  five  days.  Forty-one  days  after  the  ligature  was  tied  blood 
was  taken  from  the  second  rabbit  and  its  serum  (five  c.c.  per  kilo) 
was  injected  into  another  healthy  rabbit  whose  urine  at  that  time  did 
not  contain  albumin,  which,  however,  appeared  in  large  quantity 
immediately  after  the  injection,  and  continued  for  four  days,  rapidly 
diminishing.  The  presence  of  a  large  amount  of  albumin  in  the 
urine  of  this  animal  for  three  days  after  the  injection  led  the  investi- 
gator to  kill  the  animal  and  make  a  post-mortem  examination.  This 
was  done  on  the  seventh  day  after  the  injection.  The  kidneys  were 
found  slightly  increased  in  volume  ;  section  showed  slight  hyperemia ; 
and  the  capsule  was  easily  removed.  Microscopical  examination 
revealed  the  presence  of  marked  lesions  in  every  part  of  the  organ. 
The  vessels  of  the  glomeruli  and  all  the  capillaries  of  the  intersti- 
tial spaces  were  greatly  distended.  The  epithelium  of  the  convo- 
luted tubules  showed  necrosis,  vacuolization,  and  modifications  in  the 
nuclei ;  while  the  tubules  themselves  were  filled  with  casts.  The 
nuclei  of  the  epithelial  cells  were  irregular  and  more  or  less  broken. 
The  epithelium  of  the  straight  tubules  was  less  altered ;  but  in  these 
tubules  there  were  casts  consisting  of  epithelial  cells  so  completely 
broken  down  as  to  be  devoid  of  structure.  In  general,  the  patho- 
logical conditions  found  were  similar  to  those  which  exist  in  diffuse 
inflammation  of  the  kidney  when  accompanied  by  the  presence  of  a 
large  quantity  of  albumin  in  the  urine.  These  experiments  demon- 
strate that  the  blood  of  animals  in  which  one  ureter  has  remained 
tied  for  some  time  becomes  laden  with  a  nephrotoxic  substance. 
Evidently  ligature  of  the  ureter  is  followed  by  absorption  into  the 
circulation  of  certain  specific  substances  which  have  a  destructive 
action  upon  renal  cells.  What  the  nature  of  the  nephrotoxin  is  has 
not  been  determined,  but  the  above-mentioned  investigation  shows 
that  it  is  an  isotoxin. 

Landsteiner  ^  placed  the  spermatozoa  of  the  bullock  and  of  guinea- 
pigs  in  the  peritoneal  cavities  of  guinea-pigs ;  and  removing  them 
*  Centrcdblattf.  Bakteriologie,  25. 


i 


THE  LYSINS.  145 

from  time  to  time  and  submitting  them  to  examination,  he  ascer- 
tained that  while  the  homologous  cells  were  not  altered  and  still 
retained  their  motility,  the  heterologous  cells  soon  lost  their 
motility. 

Metschnikoff^  injected  the  spermatic  fluid  of  one  guinea-pig 
into  the  peritoneal  cavity  of  another,  and  found  that  after  some 
hours  the  spermatozoids  were  taken  up  by  the  leucocytes.  After 
this,  he  injected  into  the  peritoneal  cavities  of  guinea-pigs  sperma- 
tozoa of  several  species  of  animals,  and  observed  that  many  of  these, 
while  still  alive,  are  taken  up  by  the  leucocytes.  The  head  of  the 
spermatozoon  is  first  absorbed  by  the  leucocyte,  and  for  some  time 
thereafter  the  tail  continues  motile.  Finally,  however,  the  tail,  as 
well  as  the  head,  is  taken  into  the  leucocyte,  and  is  apparently  di- 
gested. Twenty-four  hours  after  the  injection  heads  of  spermatozoa 
within  the  leucocytes  become  more  rare,  and  finally  none  can  be  seen. 
The  majority  of  the  spermatozoa  are  taken  up  and  digested  by  the 
mononuclear  leucocytes.  Metschnikoff  was  not  able  to  observe  any 
digestion  of  spermatozoa  outside  of  the  leucocytes,  and  it  may  be 
stated  here  that  no  one  has  as  yet  prepared  a  fluid  which  dissolves 
spermatozoa.  As  we  shall  see  later,  sera  have  been  obtained  which 
destroy  the  motility  of  spermatozoa,  but  the  specific  substances  con- 
tained in  these  sera  are  spermotoxins  rather  than  spermotolysins. 
In  pursuing  his  investigations  Metschnikoff"  found  that  after  all  of 
the  injected  spermatozoa  had  been  absorbed  from  the  peritoneal 
cavity  of  the  guinea-pig,  that  animal  furnished  a  serum  which  im- 
mobilized the  spermatozoa  of  the  animal  furnishing  the  material  with 
which  it  had  been  treated.  Later,  by  the  subcutaneous  treatment  of 
rabbits  with  spermotoxic  serum  obtained  from  guinea-pigs,  he  pro- 
duced anti-spermotoxic  substances  and  found  that  spermatozoa  re- 
tained their  motility  when  placed  in  mixtures  of  spermotoxic  and 
anti-spermotoxic  sera.  This  anti-body  may  be  developed  in  castrated 
as  well  as  in  normal  animals,  showing  that  other  cells  of  the  body  may 
produce  the  substance.  Moxter  ^  injected  the  spermatozoa  of  the  ram, 
suspended  in  physiological  salt  solution,  into  the  peritoneal  cavities 
of  guinea-pigs.  After  twenty-five  minutes,  the  spermatozoa,  in  part 
still  motile,  were  seized  upon  by  the  leucocytes,  and  after  twenty- 
four  hours  no  free  spermatozoa  could  be  found.  There  was  observed 
at  no  time  any  evidence  of  dissolution  of  the  spermatic  cells.  This 
phenomenon,  it  will  be  seen,  is  quite  contrary  to  that  observed  on  the 
introduction  of  erythrocytes  into  the  peritoneal  cavity  of  an  animal 
of  another  species.  The  serum  of  the  treated  guinea-pig  was  found 
to  possess  marked  spermatocidal  properties.  When  the  living  sper- 
matozoa of  the  ram  were  divided  into  two  portions,  one  of  which  was 
treated  with  the  serum  of  a  normal  guinea-pig  and  the  other  with 

^  Annales  de  V  Institut  Pasteur,  1899. 
*  Deutsche  med.  WocfienschHft,  1900. 
10 


146  THE  LYSINS. 

the  serum  of  the  immuDized  animal,  no  marked  difference  in  the 
action  of  these  sera  could  be  observed.  In  both  instances  the  mo- 
tility of  the  cells  was  arrested  within  from  two  to  six  minutes,  when 
the  serum  was  fresh.  (It  will  be  noted  that  this  observation  is  not 
in  accord  with  that  reported  by  Metschnikoff.)  The  death  of  the 
spermatozoa  in  the  serum  is  not  due  to  the  fact  that  the  latter  does 
not  afford  an  adequate  medium  for  the  support  of  life,  because  the 
same  cells  retain  their  motility  for  four  hours  when  suspended  in 
physiological  salt  solution.  Death  is  due  to  a  toxic  substance  which 
exists  in  the  sera  of  both  normal  and  treated  animals.  This  was 
shown  to  be  the  case  by  the  following  experiment :  When  ram's 
spermatozoa  were  mixed  with  the  serum  of  either  normal  rabbits  or 
immunized  rabbits  and  the  mixture  allowed  to  stand  for  one  hour  at 
37°  and  centrifuged,  the  supernatant  fluid  was  found  to  have  lost  its 
property  of  killing  spermatozoa.  A  like  result  was  obtained  by 
heating  either  normal  serum  or  immune  serum  to  58°.  However, 
when  immune  serum  was  mixed  with  the  ram's  spermatozoa  and  the 
mixture  placed  in  the  peritoneal  cavity  of  a  normal  guinea-pig  it  was 
found  that  the  cells  were  more  speedily  killed  than  when  a  like  ex- 
periment was  made  using  the  serum  of  a  normal  rabbit.  Both  im- 
mune and  normal  sera  rendered  inactive  by  heat  were  regenerated 
when  placed  in  the  peritoneal  cavity  of  guinea-pigs,  but  after  being 
thus  regenerated,  the  immune  serum  acted  much  more  intensely  than 
the  normal  serum.  Both  immune  and  normal  sera  were  also  ob- 
served to  agglutinate  the  spermatozoa,  the  former  acting  much  more 
promptly  than  the  latter.  It  was  also  found  that  the  immune  serum 
agglutinated  the  spermatozoa  of  the  ram  after  these  cells  had  been 
deprived  of  life  by  being  kept  at  a  temperature  of  58°  for  half  an 
hour,  while  normal  serum  did  not  have  this  effect.  The  serum  of 
the  rabbit  immunized  against  the  spermatozoa  of  the  ram  did  not 
agglutinate  the  spermatozoa  of  the  horse  or  bull  and  therefore  the 
reaction  may  be  called  a  specific  one.  The  serum  of  a  rabbit  im- 
munized against  the  spermatozoa  of  the  ram  was  found  to  have  a 
marked  hemolytic  effect  on  the  blood  corpuscles  of  the  latter  animal, 
while  it  had  no  such  effect  upon  the  erythrocytes  of  the  horse  or  ox. 
The  question  arose  as  to  whether  the  spermatocidal  and  the  hemo- 
lytic action  of  the  immune  serum  were  due  to  one  or  two  substances. 
In  attempting  to  answer  this  question,  it  was  found  that  when  a 
mixture  of  spermatozoa  and  erythrocytes  from  the  ram  was  added  to 
the  immune  serum  from  the  rabbit,  the  hemolytic  action  of  the  serum 
was  arrested.  In  another  experiment  it  was  ascertained  that  when 
the  immune  serum  was  treated  with  an  excess  of  spermatozoa  and 
the  mixture  allowed  to  stand  for  some  time  at  37°,  then  centrifuged, 
the  supernatant  fluid  had  no  hemolytic  action.  From  these  observa- 
tions it  was  concluded  that  the  spermatocidal  and  the  hemolytic  sub- 
stance in  the  immune  serum  is  one  and  the  same  substance,  and  that 


THE  LYSINS.  147 

it  has  greater  affinity  for  the  spermatozoon  than  it  has  for  the  blood 
corpuscle.  Furthermore,  it  was  observed  that  when  immune  serum 
obtained  from  a  rabbit  treated  with  the  spermatozoa  of  the  ram  was 
mixed  with  the  spermatozoa  of  the  horse,  allowed  to  stand,  and  cen- 
trifuged,  the  supernatant  fluid  still  manifested  its  hemolytic  effect  on 
the  erythrocytes  of  the  ram.  Moxter  closed  his  paper  with  the  fol- 
lowing statements : 

(1)  The  immune  body  kills  the  spermatozoa  within  the  animal 
organism,  but  is  inactive  outside.  There  is  no  dissolution  of  the 
spermatozoa.  (2)  The  immune  serum  possesses  a  specific  hemolytic 
action  on  the  red  blood  corpuscles  of  the  ram  ;  it  combines  with 
both  the  spermatozoa  and  the  erythrocytes,  but  with  the  former  with 
greater  avidity.  (3)  The  immune  serum  has  a  marked  specific  ag- 
glutinating action  on  the  spermotozoa  of  the  ram.  (4)  The  erythro- 
cytes of  the  ram  are  also  agglutinated  by  the  immune  serum. 
This  action,  however,  can  be  observed  only  when  the  hemolytic  sub- 
stance has  been  rendered  inactive.  (5)  The  serum  of  a  normal  rab- 
bit also  kills  the  spermatozoa  of  the  ram,  but  acts  less  rapidly  and 
less  energetically  than  immune  serum.  The  spermatocidal  constitu- 
ent of  normal  serum  is  identical  with  the  hemolytic. 

Metalnikoff^  has  made  an  important  contribution  to  our  knowl- 
edge not  only  of  spermotoxins,  but  of  cytotoxins  in  general.  In 
some  particulars  his  investigations  contradict  those  of  Moxter. 
The  following  is  a  condensed  statement  of  his  results :  If  a  rabbit 
be  treated  with  a  mixture  of  the  spermatozoa  and  the  defibrinated 
blood  of  a  guinea-pig,  the  serum  of  the  former  animal  will  be  found 
to  be  possessed  of  marked  spermotoxic  and  hemolytic  action.  This 
serum  is  divided  into  two  portions,  numbers  1  and  2.  To  No.  1 
there  is  added  a  large  quantity  of  red  corpuscles ;  the  mixture  is 
allowed  to  stand  at  37°  for  two  hours  and  then  centrifuged.  The 
supernatant  fluid  does  not  dissolve  more  corpuscles  when  added  even 
after  the  addition  of  more  alexin  in  the  form  of  the  serum  of  a 
normal  guinea-pig.  This  shows  that  all  of  the  hemotoxic  sensitizer 
(intermediary  body)  is  fixed,  but  it  immobilizes  spermatozoa  as  read- 
ily as  it  did  before  it  was  saturated  with  red  globules.  This  shows 
that  the  spermotoxic  sensitizer  remains  intact  in  the  serum  and 
is  not  combined  with  the  red  corpuscles.  To  portion  No.  2  of 
the  serum  there  is  added  an  excess  of  spermatozoa ;  the  mixture 
is  allowed  to  stand  for  two  hours  at  37°  and  is  then  centri- 
fuged. The  supernatant  fluid  obtained  in  this  case  has  neither  sper- 
motoxic nor  hemolytic  action.  This  shows  that  the  spermatozoa  have 
combined  with  not  only  the  spermotoxic  sensitizer,  but  also  with 
the  hemotoxic.  While  the  red  corpuscles  fixed  only  the  hemotoxic 
sensitizer,  the  spermatozoa  fixed  both  sensitizers.  These  investiga- 
tions show  that  Moxter  fell  into  error  when  he  concluded  that  the 
1  Annales  de  I'Institut  Pasteur,  1900. 


148  THE  LYSINS. 

hemolytic  and  spermotoxic  action  of  the  immune  serum  was  due  to 
one  and  the  same  substance. 

Both  Metschnikoff  and  Metalnikoff  have  obtained  spermotoxic 
sera  which  are  active  both  in  vitro  and  in  vivo,  and  they  have  also 
prepared  anti-spermotoxins.  In  Metschnikoff's  experiments  the 
spermotoxic  serum  of  a  guinea-pig  was  injected  into  a  rabbit  and 
from  the  latter  there  was  obtained  an  anti-spermotoxic  serum.  After 
three  injections,  the  serum  of  this  rabbit  neutralized  the  spermotoxic 
serum  of  the  guinea-pig  in  the  proportion  of  eight  to  one.  Metal- 
nikoff proceeded  in  an  inverse  way.  He  injected  the  spermotoxic 
serum  of  a  rabbit  into  guinea-pigs,  both  subcutaneously  and  intra- 
peritoneally.  After  three  injections,  he  obtained  a  feeble  anti-spermo- 
toxic serum,  from  twenty  to  twenty-five  volumes  of  which  neutralized 
one  volume  of  spermotoxic  serum.  He  continued  to  give  injections  of 
the  spermotoxic  serum  with  the  hope  of  obtaining  a  more  active 
anti-spermotoxin,  but  instead  of  reenforcing  his  product  he  found 
that  finally  he  obtained  a  serum  which  had  absolutely  no  antitoxic 
property.  He  could  explain  this  only  on  the  supposition  that  while 
his  spermotoxic  serum  contained  two  toxic  factors,  the  sensitizer  and 
the  alexin,  his  anti-spermotoxic  serum  contained  an  anti-body  for 
only  one  of  these  factors.  For  the  neutralization  of  the  sensitizer 
there  must  be  produced  an  anti-sensitizer,  while  for  the  neutraliza- 
tion of  the  alexin  an  anti-alexin  is  needed.  On  continuing  his  in- 
vestigations along  the  line  suggested  by  this  theory  he  ascertained 
that  his  anti-spermotoxic  serum,  which  had  been  obtained  from  ani- 
mals repeatedly  treated  with  the  spermotoxic  serum  and  which  had 
shown  no  antitoxic  action,  became  active  on  the  addition  of  fresh 
serum  from  a  normal  guinea-pig ;  for  instance,  one  drop  of  the 
spermotoxic  serum  plus  ten  drops  of  the  antitoxic  serum  heated  to 
56°  plus  three  drops  of  the  serum  of  a  normal  guinea-pig  killed 
spermatozoa  within  ten  minutes ;  while  one  drop  of  the  spermotoxic 
serum  plus  ten  drops  of  the  antitoxic  serum  heated  to  56°  plus  three 
drops  of  the  serum  of  a  normal  guinea-pig  heated  to  56°,  did  not 
kill  spermatozoa.  This  shows  that  while  the  treatment  of  the  ani- 
mals with  spermotoxic  serum  produced  an  anti-sensitizer,  it  did  not 
produce  an  anti-alexin. 

Metalnikoff  has  shown  that  auto-spermotoxins  may  be  produced 
in  the  animal  body.  If  a  guinea-pig  be  treated  either  intraperito- 
neally  or  subcutaneously  with  the  spermatozoa  of  another  guinea- 
pig,  the  serum  of  the  treated  animal  is  toxic  for  the  spermatozoa  of 
both.  This  auto-toxicity  may  be  increased  by  repeating  the  injec- 
tions and  there  may  be  obtained  a  serum  ten  drops  of  which  added 
to  one  drop  of  the  spermatic  fluid  of  either  animal  causes  complete 
immobilization  of  the  spermatozoa  within  from  three  to  four  minutes. 
This  autotoxin  conforms  to  the  general  laws  of  the  cytotoxins.  It 
loses  its  toxic  property  on  being  heated  to  55°  and  is  easily  regen- 


THE  LYSINS.  149 

erated  on  the  addition  of  the  serum  of  a  normal  guinea-pig.  In  this 
connection  some  very  interesting  observations  are  recorded.  A 
guinea-pig  whose  blood  contains  a  toxin  which  is  markedly  toxic  to 
its  own  spermatozoa  in  vitro,  has  spermatozoa  which  are  quite  as 
motile  as  those  of  a  normal  animal  and  which  when  introduced  inta 
physiological  salt  solution  live  for  a  long  time  ;  however,  if  these 
spermatozoa  be  placed  in  the  serum  of  a  normal  guinea-pig  they  die 
within  from  ten  to  twenty  minutes,  while  the  spermatozoids  of  a 
normal  guinea-pig  live  in  the  serum  of  another  normal  pig  for  many 
hours.  It  is  evident  from  these  observations  that  these  spermatozoa 
have  been  acted  upon  by  the  sensitizer  circulating  in  the  blood  of 
the  guinea-pig,  but  they  are  not  killed  by  the  autotoxin  of  the  animal 
because  the  blood  does  not  contain  a  free  alexin,  this  factor  existing 
only  in  the  interior  of  the  leucocytes.  This  explains  why  autotoxins 
are  active  in  vitro  but  inactive  in  vivo.  The  cellular  elements  are 
destroyed  in  the  serum  in  vitro  and  therefore  this  fluid  contains  both 
the  sensitizer  and  the  alexin  in  the  free  state ;  while  in  the  living 
organism  all  the  cellular  elements  are  intact,  and  the  alexins  are  held 
within  the  leucocytes,  the  sensitizer  only  circulating  in  the  blood  and 
fixing  itself  to  the  spermatozoa  in  the  living  animal.  This  view  is 
supported  by  the  following  observation  :  If  one  injects  into  the  peri- 
toneal cavity  of  a  guinea-pig,  whose  blood  is  autotoxic,  living  sper- 
matozoa suspended  in  physiological  salt  solution,  and  examines  the 
peritoneal  exudate  during  the  next  five  minutes,  it  will  be  seen  that 
immediately  after  the  injection  there  is  a  rapid  phagolysis  and  that 
the  spermatozoa  do  not  die  until  the  leucocytes  break  down,  after 
which  they  are  almost  instantly  killed.  The  picture  is  wholly  dif- 
ferent, if  the  same  guinea-pig  be  treated  with  an  injection  of  physio- 
logical salt  solution  or  a  small  quantity  of  leucotoxic  serum.  In 
this  case  the  phagolysis  is  very  insignificant  after  the  injection  of  the 
living  spermatozoids  into  the  cavity.  Phagocytosis  is  established 
very  rapidly  and  soon  the  spermatozoids  while  still  alive  are  seized 
upon  by  leucocytes.  Attempts  were  made  by  Metalnikoff  to  obtain 
an  anti-auto-spermotoxin  but  without  results. 

Metschnikoff^  has  succeeded,  by  inoculating  guinea-pigs  with 
emulsions  of  the  spleen  and  mesenteric  glands  of  rats,  in  obtaining 
from  the  former  a  serum  which  agglutinates  and  dissolves  the  leu- 
cocytes of  the  latter.  The  active  agent  in  sera  thus  prepared  is 
known  as  a  leucolysin.  Funck  ^  has  produced  leucolytic  sera  by 
treating  guinea-pigs  with  emulsions  of  the  spleens  of  rabbits.  He 
began  by  injecting  half  a  spleen  at  a  time,  rubbed  up  in  physiological 
salt  solution.  After  from  eight  to  ten  days  the  injection  was  re- 
peated. From  the  animals  thus  treated  he  obtained  sera  which  dis- 
solved leucocytes  both  in  vitro  and  in  vivo.     The  alterations  brought 

'  Annales  de  P  Tnstiiut  Pasteur,  1899. 
*  Centrodblait  f.  Bakteriologie,  27. 


150  THE  LTSINS. 

about  can  be  watched  under  the  microscope  in  stained  specimens. 
The  nuclei  gradually  lose  the  stain  and  undergo  alterations  in  con- 
tour and  apparently  in  location,  seeming  to  approach  the  edge  of  the 
leucocyte.  Finally  the  cell  becomes  more  and  more  transparent 
until  it  wholly  disappears.  Serum  obtained  by  immunizing  guinea- 
pigs  to  emulsions  of  rabbit  spleen  dissolved  the  mononuclear  and 
polynuclear  cells  with  equal  rapidity,  while  the  serum  obtained  by 
inoculating  guinea-pigs  with  bone  marrow  obtained  from  rabbits  af- 
fected most  markedly  the  polynuclear  leucocytes. 

The  marked  resemblance  between  bacterial  toxins  and  digestive 
ferments  has  long  been  recognized  and  as  early  as  1893Hildebrandt^ 
immunized  animals  to  emulsin  and  diastase  and  demonstrated  the 
anti-fermentative  action  of  their  sera  both  in  vitro  and  in  vivo.  He 
injected  into  rabbits  solutions  of  diastase  and  showed  that  the  blood 
taken  from  these  animals  had  an  anti-ferment  action.  The  strength 
of  the  antitoxic  bodies  obtained  by  him  was  not  great  and  he  was 
able  to  secure  only  partial  neutralization  of  the  ferment  in  many  in- 
stances. He  rendered  a  dog  immune  to  emulsin  by  frequent  sub- 
cutaneous injections  ;  then  produced  in  this  animal  a  phloridzin  dia- 
betes and  fed  the  animal  upon  meat  and  starches.  He  found 
that  there  was  no  marked  thirst  and  the  amount  of  urine  eliminated 
was  not  increased  above  the  normal  and  the  per  cent,  of  sugar  con- 
tained in  the  urine  was  small,  while  companion  animals  in  which 
diabetes  had  been  induced  in  the  same  manner  showed  great  thirst, 
eliminated  a  large  amount  of  urine  and  excreted  large  quantities  of 
sugar.  Von  Dungern  ^  produced  anti-enzymes  by  treating  animals 
with  bacterial  proteolytic  enzymes  and  demonstrated  the  action  of 
the  anti-body  in  tubes  of  gelatin.  Gheorghiewski  ^  immunized 
animals  to  the  bacillus  pyocyaneus  and  found  that  the  serum  of  these 
animals  added  to  cultures  of  this  bacillus  prevented  the  formation 
of  the  blue  coloring  matter  characteristic  of  the  germ  and  the  for- 
mation of  which  is  dependent  upon  the  presence  of  pepton.  Mor- 
genroth  *  immunized  goats  against  rennet  and  obtained  in  the  serum 
of  these  animals  a  fairly  active  anti-rennet  body.  The  specimen  of 
rennet  which  he  used  in  inducing  the  immunization  coagulated  milk 
when  added  to  it  in  the  proportion  of  1  :  3,000,000  ;  the  strongest 
anti-body  that  he  could  obtain  when  added  to  milk  to  the  extent 
of  two  per  cent.,  prevented  the  action  of  rennet  when  added  in  the 
proportion  of  1  :  20,000,  while  1  :  15,000  still  induced  coagulation. 
From  his  studies  he  concludes  that  rennet  resembles  the  bacterial 
toxins  and  possesses  a  haptophorous  and  a  zymophorous  group,  to  the 
latter  of  which  the  specific  action  of  the  ferment  is  due,  and  it  is 

'  Virchow's  Archiv,  131. 

^  Munchener  med.   Wochenschrift,  1898,  1040. 

^  AuTiales  de  I' Institut  Pasteur,  1899. 

*  CentrcUblatt  f.  Bakteriologie,  26. 


THE  LYSINS.  151 

against  this  group  that  the  anti-body  manifests  its  action.  He  also 
showed  that  the  milk  of  the  immunized  goat  contains  the  anti-body 
and  does  not  coagulate  on  the  addition  of  rennet  in  quantities  which 
are  sufficient  to  coagulate  the  milk  of  normal  animals.  Buchner  ^ 
thinks  that  the  alexin  (the  complement,  according  to  Ehrlich's 
theory)  is  a  ferment  and  that  it  is  one  and  the  same  body  in  the 
blood  of  all  species  of  animals.  The  intermediary  body  (according 
to  Ehrlich's  theory)  is  the  only  substance  against  which  immunity 
can  be  obtained.     This  Buchner  believes  to  be  specific. 

^  Munchener  med.  Wochenschrift,  1900. 


CHAPTER  VIII. 

THE   AGGLUTININS. 

In  1889  Charrin  and  Roger  ^  in  studying  the  action  of  the  serum 
of  sick  and  immunized  animals  on  homologous  bacteria,  observed 
that  the  bacillus  pyocyaneus  behaved  peculiarly  when  placed  in  the 
serum  of  an  animal  which  had  been  immunized  to  this  organism. 
In  the  serum  of  a  normal  rabbit,  this  germ  was  found  to  grow  as  it 
does  in  beef  tea,  or  other  liquid  nutritive  medium,  forming  an 
opaque  culture,  while  in  the  serum  of  an  immunized  rabbit  these 
microbes  were  found  to  form  small  floating  floccules  which  soon  sub- 
sided, leaving  a  clear,  supernatant  fluid.  It  was  furthermore  ob- 
served that  the  germ  grown  in  its  homologous  serum  does  not  pro- 
duce pyocyanin,  while  in  the  serum  of  normal  animals  this  coloring 
matter  is  formed  by  the  growth  of  the  bacillus.  The  sera  of  ani- 
mals sick  from  inoculation  with  the  bacillus  pyocyaneus,  and  of 
those  immunized  against  this  germ,  were  observed  to  have  the  same 
effect  on  its  growth.  This  early  observation  indicated,  as  has  been 
subsequently  more  clearly  shown,  that  the  phenomenon  of  aggluti- 
nation is  a  reaction  of  infection  as  well  as  of  immunization. 

In  his  studies  on  immunity,  MetchnikofP,^  found  that  the  vibrio 
which  bears  his  own  name  behaves  in  a  manner  similar  to  that  ob- 
served by  Charrin  and  Roger  with  the  bacillus  pyocyaneus.  On 
this  point  Metschnikoff"  makes  the  following  statement :  "  In  the 
blood  and  serum  of  non-vaccinated  guinea-pigs  the  vibrio  develops 
as  it  does  in  ordinary  liquid  media,  the  individual  organisms  retain- 
ing their  motility  and  remaining  distinct  one  from  the  other.  On 
the  other  hand  in  the  blood  and  serum  of  vaccinated  animals  the 
vibrios  become  immobile,  and  form  smaller  or  larger  floccules  which 
float  in  the  liquid." 

In  1893  Issaeif  ^  observed  a  like  phenomenon  when  the  pneumo- 
coccus  was  placed  in  its  homologous  serum,  and  this  observation  was 
repeated  later  in  the  studies  on  immunity  prosecuted  by  the  same 
author  in  conjunction  with  Ivanoff.*  In  the  last-mentioned  com- 
munication there  is  found  the  following  statement :  "  In  the  blood 
serum  of  healthy,  non-immunized  guinea-pigs  the  vibrio  develops 
rapidly,  and  after  from  four  to  five  hours  at  37°  there  is  a  uniform 

'  Comptes  JRendus  de  I'  Academic  des  Sciences,  109. 
^Annates  de  PlnstitM  Pasteur,  5,  1891. 
'  Annates  de  I'  Institut  Pasteur,  7. 
*  Zeitschrift  fUr  Hygiene,  17. 

152 


THE  AGGLUTININS.  153 

cloudiness  throughout  the  fluid,  while  the  surface  is  covered  by  a 
scum ;  but  in  immune  serum  the  microbes  sink  to  the  bottom  of  the 
tube,  while  the  supernatant  fluid  remains  clear.  This  condition  con- 
tinues for  from  eight  to  nine  days,  and  it  is  not  until  the  tenth  day 
that  the  culture  becomes  cloudy  and  a  scum  appears  on  the  surface." 
While  the  above-mentioned  observations  were  made  as  herewith 
stated,  they  attracted  no  special  attention,  and  in  all  probability 
their  significance  was  not  appreciated  by  the  observers  themselves, 
and  our  knowledge  of  the  phenomenon  of  agglutination  may  be  said 
to  have  originated  in  the  classical  research  of  Gruber  and  Durham,^ 
which  was  reported  in  1896.  These  investigators  found  that  when 
a  suspension  of  an  agar  culture  of  the  cholera  or  typhoid  bacillus  is 
mixed  with  its  homologous  serum,  the  bacteria  lose  their  motility  and 
form  large  clumps  or  masses.  They  attributed  this  phenomenon  to 
the  presence  of  specific  substances  in  the  serum,  for  which  they  pro- 
posed the  term  agglutinins,  and  suggested  that  the  phenomenon 
might  be  designated  that  of  agglutination.  Their  researches  showed 
that  this  reaction  is  specific,  within  certain  limitations :  For  instance, 
a  cholera  serum  has  no  agglutinating  effect  upon  the  vibrio  of  Fink- 
ler,  that  of  Metschnikoff  or  that  of  Rumpel,  and  typhoid  serum  is 
without  action  upon  any  of  the  colon  germs,  but  a  cholera  serum 
does  agglutinate,  v.  Ivanoff,  v.  Berolinensis  and  v.  Seine- Versailles. 
In  all  instances  in  which  cholera  serum  was  found  to  have  an  agglu- 
tinating action,  it  was  also  found  to  protect  animals  against  inocula- 
tion with  the  germ,  and  these  two  properties  vary  in  the  same  pro- 
portion, that  is,  the  greater  the  agglutinating  action  the  greater  is 
the  protective  power  against  the  same  germ.  Typhoid  serum  was 
found  to  agglutinate  typhoid-like  bacilli,  which  were  positively 
known  not  to  be  real  typhoid  germs.  For  instance,  bacillus  enter- 
itidis  is  agglutinated  in  a  typical  manner  by  a  highly  active  typhoid 
serum.  However,  there  is  easily  observable  a  quantitative  difference 
in  the  agglutinating  action  of  both  cholera  and  typhoid  sera  upon 
other  than  their  homologous  bacteria.  Moreover,  the  agglutinating 
action  of  typhoid  serum  is  apparently  more  strictly  specific  than  is 
that  of  cholera  serum,  but  in  making  a  practical  test  with  either,  as 
we  shall  see  later,  dilution  of  the  serum  is  necessary  in  order  to 
avoid  error.  Gruber  and  Durham  also  pointed  out  the  fact  that 
normal  sera  may  have  a  more  or  less  marked  agglutinating  action 
upon  both  the  vibrio  of  Asiatic  cholera  and  the  bacillus  of  typhoid 
fever,  and  they  stated  that  in  order  to  use  this  reaction  for  the  pur- 
pose of  identifying  bacteria  it  would  be  found  to  be  necessary  to  dilute 
the  serum  employed.  It  seems  to  have  been  in  the  mind  of  these  in- 
vestigators at  the  time  of  the  publication  of  their  first  paper  that  the 
reaction  described  by  them  would  find  its  practical  application  in  the 
detection   and   the  definite  recognition  of  specific  microorganisms 

^  Munchener  med.   Wochenschrift,  43. 


154  THE  AGGLUTININS. 

rather  than  in  the  diagnosis  of  disease.  They  recommended  that 
large  guinea-pigs  should  be  immunized  by  intraperitoneal  injections 
of  dead  cultures,  continued  for  from  four  to  six  weeks,  until  a  high 
degree  of  immunization  should  be  secured,  and  that  the  peritoneal 
lymph  or  blood  serum  of  these  animals  might  be  used  to  determine 
with  certainty  whether  or  not  a  suspected  microorganism  was  the 
vibrio  of  Asiatic  cholera  or  the  bacillus  of  typhoid  fever.  They 
stated  that  if  with  such  a  serum  from  an  animal  immunized  with  the 
cholera  germ  agglutination  does  not  occur  promptly  and  completely, 
it  can  be  said  with  certainty  that  the  germ  under  suspicion  is  not 
the  vibrio  of  cholera,  and  the  same  test  can  be  applied  with  equal 
certainty  in  the  detection  of  the  typhoid  bacillus.  On  the  other 
hand,  if  the  reaction  is  positive,  the  identity  of  the  microorganism 
must  still  remain  in  doubt.  We  have  carefully  read  the  original 
paper  of  Gruber  and  Durham,  and  we  fail  to  find  therein  any  reason 
for  believing  that  these  investigators  at  that  time  had  any  idea  that 
the  phenomenon  which  they  were  investigating  was  soon  to  become 
one  of  the  most  certain  and  easily  applicable  methods  for  the  diag- 
nosis of  typhoid  fever.  It  is  true  that  they  speak  of  the  serum  test 
as  a  diagnostic  measure,  but  from  the  context  it  appears  that  by  the 
term  diagnosis  they  mean  the  specific  and  positive  identification  of  a 
suspected  bacterium. 

A  few  months  after  the  publication  of  the  last-mentioned  contri- 
bution, Widal  ^  reported  the  successful  application  of  the  phenomenon 
of  agglutination  in  the  diagnosis  of  typhoid  fever,  and  among  clini- 
cians it  is  generally  known  as  the  "  Widal  reaction." 

Our  scientific  information  concerning  the  agglutinins  has  been  ob- 
tained principally  from  the  researches  of  Widal  and  Sicard,^  NicoUe,' 
and  Winterberg.* 

It  is  quite  evident  that  in  such  a  reaction  as  that  observed  in  ag- 
glutination there  must  be  two  factors.  One  of  these,  which  we  may 
designate  the  agglutinin,  is  found  in  the  serum ;  while  the  other, 
which  may  be  denominated  the  agglutinable  substance,  is  furnished 
by  the  bacterial  culture.  The  product  which  results  from  the  reac- 
tion between  these  bodies  may  be  designated  the  agglutinate.  We 
will  first  give  our  attention  to  the  agglutinins.  The  normal  serum 
of  certain  animals  is  capable  of  manifesting  a  slight  agglutinating 
action  upon  certain  bacteria,  notably  the  colon  bacillus  and  the  ba- 
cillus of  Eberth.  The  normal  serum  of  the  horse,  donkey,  dog,  and 
rabbit,  when  added  to  cultures  of  the  Eberth  bacillus  without  dilu- 
tion causes  agglutination.  Indeed,  the  serum  of  these  animals  may 
agglutinate  the  typhoid  bacillus  when  diluted  1  to  30.  The  nor- 
mal serum  of  the  guinea-pig  has  no  agglutinative  action  on  this  or- 

'  Societe  MedAcah  des  Hospitaux,  June  26,  1896. 
'^Annales  de  P  Institut  Pasteur,  11. 
'  Annalts  de  V Institut  Pasteur,  8. 
*  Zeits.  fixr  Hygiene,  32. 


THE  AGGLUTININS.  155 

ganism.  The  serum  of  men  who  have  never  had  typhoid  fever 
may,  when  the  dilution  is  not  greater  than  one  to  ten,  agglutinate 
the  Eberth  bacillus,  and  it  should  be  remarked  that  the  manifesta- 
tion of  this  property  by  the  sera  of  different  individuals  varies 
within  wide  limits.  It  is  still  a  question  whether  the  agglutinating 
action  manifested  in  the  serum  of  immunized  animals,  or  that  of 
persons  sick  with  typhoid  fever,  is  due  to  an  exaggeration  of  a  nor- 
mal property,  or  is  caused  by  the  presence  of  a  new  substance. 

The  period  in  the  course  of  typhoid  fever  when  marked  aggluti- 
nation is  first  manifested  by  the  serum  varies  widely  in  different 
cases.  As  a  rule,  agglutination  in  a  well-marked  form  is  not  ob- 
tained until  after  the  seventh  day,  but  there  are  cases  on  record  in 
which  it  has  been  obtained  as  early  as  the  second  day,  and  there  are 
others  in  which  it  has  been  delayed  until  the  second,  third,  and  even 
fourth  week.  Likewise  the  disappearance  of  the  specific  agglu- 
tinating property  from  the  serum  after  recovery  from  typhoid  fever 
is  very  variable  in  time.  The  reaction  has  been  known  to  fail  ten 
days  after  the  establishment  of  apyrexia,  while  in  other  instances 
marked  agglutination  has  been  secured  many  years  after  recovery 
from  typhoid  fever.  The  intensity  of  the  reaction  varies  widely, 
and  is  not  always  in  proportion  to  the  severity  of  the  disease. 
Many  methods  of  measuring  the  agglutinative  power  of  sera  have 
been  proposed.  One  recommended  by  Widal  and  Sicard  we  have 
found  to  be  satisfactory.  A  number  of  tubes  containing  each  one, 
two,  three,  four,  five,  etc.,  c.c.  of  bouillon  are  kept  on  hand.  When 
a  test  is  to  be  made,  one  adds  a  drop  of  the  serum  to  each  of  these 
tubes  and  then  inoculates  the  tube  with  a  typhoid  culture.  The 
tubes  thus  prepared  are  placed  in  the  incubator  and  kept  at  37°  for 
from  four  to  six  hours.  At  the  expiration  of  this  time  it  may  be 
seen  at  a  glance  in  which  tubes  agglutination  has  occurred,  inasmuch 
as  the  contents  of  these  tubes  will  be  unclouded  and  the  bacilli  will 
be  found  deposited  on  the  bottom.  If  this  reaction  does  not  take 
place  in  tube  number  one,  which  represents  a  dilution  of  1  to  20, 
the  test  is  considered  as  wholly  negative.  If  number  three  contains 
clumps,  while  number  four  is  uniformly  clouded,  the  serum  is  active 
in  the  proportion  of  one  to  sixty  and  inactive  in  the  proportion  of 
one  to  eighty.  An  absolute  determination  of  the  agglutinating 
property  of  a  given  serum  is  probably  not  within  the  range  of  pos- 
sibility, and  we  indicate  the  relative  activity  of  different  sera  by  the 
dilutions  in  which  the  reactions  are  observable.  The  number  of 
germs  present  certainly  has  some  influence,  and  it  is  a  well-known 
fact  that  the  age  of  the  culture  is  of  importance  in  making  this  test. 
In  young  cultures  the  bacilli  are  more  motile,  and  there  is  less  tend- 
ency to  manifest  the  phenomenon  of  pseudo-agglutination. 

Agglutinins  exist  not  only  in  the  blood,  but  in  other  fluids  of  the 
body.     The  manifestation  of  agglutinating  power  by  the  urine  is  in 


156  THE  AGGLUTININS. 

the  same  individual  variable  from  day  to  day  and  even  from  hour  to 
hour,  and  so  far  we  have  no  satisfactory  explanation  of  this  varia- 
tion. Compared  with  the  agglutinating  property  of  the  blood,  that 
of  the  urine  is  always  feeble.  This,  together  with  its  variability, 
renders  the  use  of  this  secretion  in  place  of  blood  serum  impractic- 
able. Serum  obtained  by  vesication  often  shows  marked  agglutinat- 
ing power.  In  the  few  cases  in  which  the  test  has  been  made,  bile 
manifests  this  reaction  in  a  fairly  well  marked  manner.  Often  the 
reaction  may  be  easily  and  plainly  obtained  with  tears.  However, 
this  secretion  is  subject  to  marked  variation  in  its  agglutinating 
power,  and  it  has  been  found  that  tears  obtained  by  artificial  stimu- 
lation are  generally  devoid  of  this  property.  In  immunizing  animals 
it  has  been  found  that  pus  obtained  from  abscesses  due  to  the  bacillus 
of  Eberth  may  show  a  high  degree  of  agglutinative  power.  Agglu- 
tinins may  pass  from  the  mother  to  the  fetus  and  may  be  transferred 
from  the  mother  to  the  child  in  the  milk.  The  agglutinins  seem  to 
be  fairly  stable  bodies,  and  some  of  them  may  be  heated  to  140° 
without  destruction,  but  that  of  the  typhoid  bacillus  is  destroyed  at  a 
temperature  of  115°.  Antiseptics,  so  far  as  they  have  been  tested 
seem  to  be  without  effect  upon  the  agglutinins.  Typhoid  serum 
may  be  treated  with  formalin  and  kept  quite  indefinitely  without  loss 
of  its  agglutinating  property.  However,  the  use  of  antiseptics  is  not 
necessary  in  order  to  have  a  serum  retain  its  agglutinating  action. 
A  typhoid  serum  may  be  used  as  a  culture  medium  for  the  germs  of 
cholera,  anthrax,  diphtheria,  or  of  the  ordinary  putrefactive  bacteria 
without  diminution  of  its  agglutinating  property.  Indeed,  a  highly 
active  serum  will  retain  its  agglutinative  action  after  it  has  been 
made  putrid  by  bacterial  growth.  Dilute  mineral  acids  decrease  the 
agglutinating  property  of  typhoid  sera,  but  when  the  acid  has  been 
added  in  very  dilute  form  and  has  not  remained  long  in  contact  with 
the  serum,  partial  restoration  may  be  induced  by  neutralization  with 
alkali.  Acetic  acid  has  a  like,  but  less  marked  effect.  Caustic 
alkalis  have  an  action  similar  to  that  manifested  by  acids.  Appar- 
ently the  agglutinins  are  not  altered  by  any  of  the  digestive  ferments. 
Indeed,  pepsin  seems  to  protect  agglutinins  to  some  extent  against 
the  effects  of  dilute  hydrochloric  acid,  and  trypsin  has  a  similar  pro- 
tective action  against  dilute  alkalis. 

Filtration  of  blood  serum  through  porcelain  removes  the  agglu- 
tinins, inasmuch  as  it  has  been  demonstrated  that  the  filtrate  has  lost 
largely  or  altogether  the  agglutinating  property.  Saturation  of 
typhoid  serum  with  magnesium  sulphate  leads  to  precipitation  of 
the  greater  part  or  all  of  the  agglutinin.  If  the  precipitate  be  dis- 
solved in  water  it  will  be  found  that  the  agglutinin  has  not  been 
destroyed  by  the  action  of  this  reagent.  Half  saturation  with 
ammonium  sulphate  precipitates  practically  all  of  the  agglutinin ; 
and  complete  saturation  with  chlorid  of  sodium  has  a  like  effect.     It 


THE  AGGLUTININS.  157 

may  be  inferred  from  these  reactions  that  agglutinin  is  one  of  the 
globulins  or  albumins  existing  in  the  blood.  However,  this  conclu- 
sion does  not  necessarily  follow.  It  is  altogether  within  the  range 
of  possibility  that  the  agglutinin  is  carried  down  mechanically,  as 
happens  to  certain  ferments  and  toxins  when  albuminous  solutions 
are  treated  with  neutral  salts.  The  fact  that  the  agglutinating  action 
has  been  secured  with  urine  in  which  no  albumin  or  globulin  could 
be  detected,  has  led  some  to  believe  that  the  agglutinins  belong  to 
neither  of  these  bodies.  This  conclusion  also  is  unjustifiable. 
Because  we  cannot  detect  a  globulin  or  an  albumin  in  the  urine  is 
no  positive  proof  that  one  or  both  of  these  substances  may  not  be 
present.  It  simply  means  that  neither  is  present  in  sufficient  quan- 
tity to  respond  to  the  test  employed.  It  must  follow  therefore  that 
we  are  not  yet  in  a  position  to  determine  whether  or  not  the  agglu- 
tinins belong  to  the  proteid  constituents  of  blood  serum. 

A  highly  active  serum  after  having  been  diluted  with  water  until 
no  test  for  proteids  in  it  can  be  obtained  may  still  manifest  its  ag- 
glutinating action,  while,  on  the  other  hand,  a  serum  possessed  of 
but  little  activity  loses  its  agglutinating  property  on  dilution  long 
before  positive  tests  for  the  presence  of  proteids  fail. 

Precipitation  of  typhoid  serum  with  absolute  alcohol  destroys  the 
agglutinin.  This  is  probably  due  to  the  dehydrating  action  of  alco- 
hol, because  if  as  soon  as  the  precipitate  is  formed  it  be  collected  on 
a  filter,  and  the  alcohol  allowed  to  drain  away  and  the  precipitate  ex- 
tracted with  physiological  salt  solution,  this  extract  will  be  found  to 
be  possessed  of  a  certain  amount  of  agglutinating  power,  while,  on 
the  other  hand,  if  the  precipitate  be  allowed  to  remain  for  some 
hours  under  the  alcohol,  the  agglutinin  is  wholly  destroyed. 

The  agglutinating  action  of  a  blood  plasma  obtained  by  the  addi- 
tion of  oxalate  of  potassium  to  the  blood  is  more  marked  than  that 
of  the  serum  of  the  same  blood.  It  follows  from  this  that  the  ag- 
glutinin must  be  in  part  retained  in  the  blood  clot.  Indeed,  this 
can  be  positively  demonstrated  by  testing  the  agglutinating  action  of 
an  extract  of  the  clot  made  with  physiological  salt  solution.  Widal 
and  Sicard  have  shown  quite  positively  that  the  white  blood  corpus- 
cles, outside  of  the  body  at  least,  do  not  furnish  the  agglutinins.  The 
experiment  by  means  of  which  this  demonstration  was  accomplished 
may  be  briefly  described  as  follows  :  A  sterilized  collodion  tube  was 
filled  with  typhoid  blood,  to  which  oxalate  of  potassium  had  been 
added,  and  allowed  to  stand.  The  red  corpuscles  soon  formed  a  de- 
posit at  the  bottom  of  the  tube,  and  these  were  covered  by  a  thin  coat 
of  white  corpuscles,  and  above  these  stood  the  plasma.  The  plasma 
was  removed  with  a  pipette  as  thoroughly  as  possible  without  taking 
up  any  of  the  leucocytes.  Next  the  tube  was  constricted  by  a  string 
tied  around  it  on  a  line  between  the  layers  of  white  and  red  corpus- 
cles.    After  these  portions  had  stood  for  twenty-four  hours  the  rela- 


158  THE  AGGLUTININS. 

tive  agglutinating  property  of  each  was  tested,  and  it  was  found  that 
the  plasma  containing  the  white  corpuscles  had  no  greater  action 
than  did  that  which  was  devoid  of  these  elements.  Of  course,  this 
experiment  does  not  indicate  that  the  leucocytes  have  nothing  to  do 
with  the  formation  of  agglutinins  in  the  living  body.  However, 
that  the  agglutinins  are  in  solution  in  the  circulating  blood  is  shown 
by  their  appearance  in  certain  secretions  and  exudates. 

Nicolle  reported  that  the  agglutinable  substance  is  soluble  in  ab- 
solute alcohol  and  ether.  If  this  statement  had  been  confirmed,  the 
separation  of  this  body  from  the  proteids  would  be  comparatively 
easy,  but  the  researches  of  Winterberg  demonstrate  that  Nicolle  had 
fallen  into  error  on  this  point.  The  last-mentioned  investigator 
made  the  following  experiment  bearing  on  the  question  :  A  culture  of 
the  typhoid  bacillus  in  bouillon,  which  had  grown  for  seventy  days 
at  37°  and  which  contained  a  large  number  of  germs,  was  treated 
with  ten  times  its  volume  of  absolute  alcohol.  The  precipitate  was 
extracted  first  with  absolute  alcohol  and  then  with  ether,  each  of 
which  was  decanted  after  twenty-four  hours.  Both  of  these  ex- 
tracts were  mixed  with  the  alcoholic  filtrate  obtained  after  precipita- 
tion, and  the  whole  evaporated  to  dryness  in  vacuo  at  30°.  The  resi- 
due was  taken  up  with  feebly  alkaline  bouillon  and  this  solution 
again  precipitated  with  ten  times  its  volume  of  absolute  alcohol,  and 
the  filtrate  evaporated  as  before.  The  residue  last  obtained  was 
taken  up  with  bouillon,  filtered  through  porcelain,  and  the  solution 
failed  to  manifest  any  reaction  when  mixed  with  a  highly  active 
typhoid  serum.  Winterberg  furthermore  showed  that  typhoid  ag- 
glutinin is  produced  in  animals  which  have  been  treated  with  alco- 
holic precipitates  obtained  from  both  filtered  and  unfiltered  typhoid 
cultures. 

The  fact  that  typhoid  cultures,  the  germs  of  which  have  been  de- 
stroyed by  the  use  of  disinfectants  or  the  application  of  heat,  still 
agglutinate  homologous  sera  demonstrates  that  the  agglutinable  sub- 
stance contained  in  these  cultures  is  not  destroyed  when  the  organ- 
isms are  deprived  of  life.  Widal  and  Sicard  found  that  typhoid 
bouillon  cultures  exposed  for  half  an  hour  to  a  temperature  of  from 
70°  to  100°  still  react  when  brought  in  contact  with  a  highly  potent 
serum,  but  that  the  reaction  in  this  case  is  not  so  marked  as  that  ob- 
served in  living  cultures.  The  clumps  form  more  slowly  and  are 
less  voluminous.  When  a  typhoid  culture  was  heated  for  half  an 
hour  from  57°  to  60°  and  then  brought  in  contact  with  an  active 
serum  the  reaction  was  found  to  be  apparently  identical  with  that 
obtained  with  living  cultures.  It  is  evident  from  these  experiments 
that  the  agglutinable  substance  is  not  destroyed  by  the  temperatures 
above  mentioned,  and  that  it  is  present  in  dead  as  well  as  in  living 
cultures. 

Widal  and  Sicard  showed  that  the  dried  blood  of  typhoid  patients 


THE  AGGLUTININS.  159 

can  be  used  in  making  the  agglutination  test.  They  impregnated 
small  bits  of  sponge  with  four  or  five  drops  of  the  blood,  allowed  to 
dry,  and  subsequently  macerated  the  pieces  for  half  an  hour  in  from 
ten  to  fifteen  drops  of  bouillon.  The  addition  of  this  extract  to 
bouillon  cultures  of  the  Eberth  bacillus  induced  typical  agglutination. 
Johnston  ^  first  demonstrated  that  a  drop  of  blood  allowed  to  dry 
upon  non-absorbent  paper  may  be  transported  through  any  distance 
and  kept  indefinitely  and  still  be  capable,  after  being  moistened  with 
water,  of  giving  the  agglutination  test.  In  this  way  this  method  of 
making  a  diagnosis  of  typhoid  fever  has  been  popularized  and  placed 
practically  within  the  reach  of  every  physician.  It  is  safe  to  say 
that  at  present  more  than  90  per  cent,  of  the  agglutination  tests  made 
are  applied  to  dried  blood  stains.  The  only  objection  to  this  method 
is  that  exactness  in  dilution  can  not  be  secured,  and  much  must  de- 
pend upon  the  judgment  and  experience  of  the  individual  making  the 
test.  However,  for  clinical  purposes,  dried  blood  gives,  in  a  large 
per  cent,  of  cases,  evidence  which  is  of  positive  value.  Usually  the 
blood  stain  is  rubbed  up  with  either  sterilized  water  or  physiological 
salt  solution,  and  this  is  added  to  a  bouillon  culture  of  the  typhoid 
bacillus  twenty- four  hours  old.  A  hanging  drop  of  this  mixture  is 
prepared  and  examined  under  the  microscope.  The  extent  to  which 
the  blood  or  serum  should  be  diluted  is  still  a  matter  about  which 
there  is  a  difference  of  opinion.  The  general  rule  is  that  the  dilu- 
tion should  not  be  less  than  1  to  20  and  that  agglutination  in  this 
dilution  should  occur  within  thirty  minutes.  However,  in  order 
to  be  quite  sure  concerning  the  result,  the  dilution  should  not  be  less 
than  1  to  60,  and  if  agglutination  occurs  in  this  dilution  within  thirty 
minutes,  the  evidence  that  the  blood  has  come  from  an  individual 
with  typhoid  fever  is  quite  positive.  With  dilutions  of  1  to  30  or 
less,  agglutination  is  frequently  obtained  with  normal  serum,  and 
more  frequently  given  in  certain  diseases  such  as  tuberculosis,  pneu- 
monia, septicemia,  and  influenza.  When  agglutination  does  not 
occur  in  the  dilution  of  1  to  60,  but  is  observed  in  the  dilution  of  1 
to  30  the  weight  of  evidence  is  in  favor  of  the  existence  of  typhoid 
fever,  but  the  proof  cannot  be  considered  as  positive.  In  this,  as  in 
the  application  of  many  other  clinical  tests,  much  depends  upon  the 
skill,  experience  and  good  judgment  of  the  observer.  It  appears 
that  about  96  per  cent,  of  the  cases  of  typhoid  fever  furnish  some 
time  during  the  course  of  the  disease  a  serum  which  will  give  this 
reaction.  It  will  be  seen  from  this  that  failure  to  secure  the  agglu- 
tination test  is  not  absolute  proof  of  the  non-existence  of  typhoid 
fever.  Experience  has  shown  that  attenuated  cultures  are  better 
suited  for  the  agglutination  test  than  more  virulent  ones.  However, 
the  evidence  on  this  point  is  by  no  means  unanimous,  and  an  explana- 
tion is  not  easily  given.  Young  cultures  are  better  than  older  ones, 
^  New  York  Med.  Journal,  44,  1896. 


160  THE  AGGLUTININS. 

because  motility  of  the  individual  organisms  is  greater  in  the  former, 
and  besides  the  old  cultures  may  contain  clumps  consisting  partly  of 
dead  and  disintegrated  bacterial  cells,  which  may  be  mistaken  for 
typical  agglutination.  A  like  source  of  error  is  met  with  when  the 
drop  to  be  examined  is  allowed  to  partially  dry  on  the  cover-glass. 
These  appearances,  which  may  be  mistaken,  especially  by  the  inex- 
perienced, for  true  agglutination,  have  been  designated  pseudo-re- 
actions. There  is  also  the  possibility,  especially  when  dried  blood  is 
used,  of  mistaking  undissolved  clumps  of  red  cells  containing  threads 
of  fibrin  for  the  true  clumping  of  bacilli. 

When  exact  scientific  work  is  to  be  done  it  is  better  to  have  the 
blood  drawn  directly  from  a  prick  on  the  finger  or  ear  into  a  gradu- 
ated pipette  in  which  the  dilution  can  be  made  with  exactness. 
There  are  special  tubes  made  for  this  purpose,  and  the  pipette  which 
accompanies  the  ordinary  hematocytometer  may  be  used.  In  hos- 
pital practice,  and  especially  when  a  large  amount  of  serum  is  de- 
sired, a  small  Spanish  fly  blister  may  be  used,  and  if  properly  cared 
for  need  cause  no  pain.  As  has  been  already  stated,  cultures  killed 
by  heat  or  those  preserved  by  the  addition  of  formalin,  thymol,  or 
some  other  disinfectant  may  be  used,  but  these  are  more  or  less  un- 
satisfactory, inasmuch  as  the  observer  loses  the  opportunity  of 
watching  the  gradual  decrease  of  motility. 

As  has  been  stated,  Gruber  and  Durham  probably  expected  that 
the  greatest  benefit  likely  to  come  from  their  studies  would  lie  in 
the  fact  that  this  reaction  would  furnish  a  positive  means  of  differ- 
entiating one  species  of  bacteria  from  another.  It  is  especially  de- 
sirable that  we  should  have  some  positive  and  easily  applicable 
method  of  distinguishing  between  the  colon  and  typhoid  bacilli,  and 
it  was  thought  that  the  agglutination  test  supplies  this  long-felt  want. 
However,  more  extended  observation  has  shown  that  typhoid  serum 
occasionally  agglutinates  the  colon  bacillus,  and  very  rarely  the 
agglutination  with  this  organism  may  be  more  marked  than  it  is 
with  the  typhoid  germ.  It  may  be  that  in  cases  of  this  kind  the 
typhoid  patient  is  suffering  from  a  mixed  infection  in  which  both 
the  colon  and  the  typhoid  bacilli  participate.  When  the  agglutina- 
tion test  is  used  for  the  purpose  of  identifying  a  bacterium  the  serum 
used  should  be  taken  from  an  animal  immunized  to  a  pure  culture  of 
that  microorganism,  and  reliance  for  the  differentiation  of  typhoid 
and  colon  bacilli  should  not  be  placed  in  sera  obtained  from  men 
with  typhoid  fever. 

It  must  be  plainly  evident  from  what  has  already  been  said  that 
the  agglutination  test  has  not  that  specificity  which  is  possessed  by 
the  lysins  and  precipitins.  This  was  plainly  seen  by  Gruber  and 
Durham  in  their  original  communication,  and  it  has  been  in- 
sisted upon  by  Durham  in  a  later  paper,  in  which  he  suggests  that 
it  would  be  better  to  employ  the  word  " special" 


THE  AGGLUTININS.  161 

There  have  been  proposed  several  hypotheses  concerning  the  phe- 
nomenon of  agglutination.  Gruber  suggested  that  there  must  be 
something  in  the  immune  serum  which  profoundly  alters  its  homolo- 
gous bacterium.  This  alteration  consists,  in  part  at  least,  in  in- 
creased viscosity  of  the  cell  membrane  of  the  bacterium.  The  pro- 
duction of  the  adhesive  substance  is  supposed  to  be  the  chief  factor 
in  the  agglutination  or  clumping  of  the  bacterial  cells.  When  two 
or  more  germs  are  brought  together,  either  by  active  or  passive  move- 
ment, they  adhere  one  to  the  other,  lose  their  individual  motility  and 
form  small  masses  which  gradually  subside  in  the  culture  medium. 
According  to  Nicolle,  the  agglutinin,  which  is  furnished  by  the  serum, 
precipitates  the  agglutinable  substance,  which  is  formed  within  the 
bacterial  cell,  from  which  it  diffuses  through  the  culture  medium  to 
a  greater  or  less  extent.  The  agglutinable  substance  is  supposed  to 
be  most  abundant  in  the  cell  membrane,  and  by  the  union  of  the  ag- 
glutinin and  the  agglutinable  substance  small  coagula  are  formed  and 
surround  the  bacterial  cells.  The  theory  proposed  by  Paltauf  varies 
but  slightly  from  that  suggested  by  Nicolle.  However,  the  former 
supposes  that  the  combination  between  the  agglutinin  and  the  agglu- 
tinable substance  occurs  wholly  outside  of  the  bacterial  cells  aud  that 
the  microorganisms  are  involved  in  the  floccules  mechanically  only. 
The  hypothesis  of  Dineur  varies  from  that  originally  proposed  by 
Gruber  only  by  transferring  the  formation  of  the  adhesive  matter 
from  the  bacterial  cell  walls  to  the  cilia.  Diminished  motility  and 
subsequent  agglutination  are  explained  on  the  supposition  that  the 
cilia  are  so  altered  that  they  secrete  an  adhesive  substance.  In  1896 
Bordet  suggested  a  hypothesis  in  which  he  attempted  to  explain  the 
phenomenon  of  agglutination  wholly  upon  physical  grounds.  He 
suggested  that  in  homologous  sera  there  is  present  a  substance  or 
substances  which  change  the  relative  molecular  attraction  between 
the  microbes  and  the  ambient  fluid.  Later  Bordet  developed  this 
theory  as  we  have  already  indicated  in  our  discussion  of  the  lysins. 
In  accordance  with  the  general  trend  of  French  thought  along  these 
lines,  he  has  rejected  the  German  idea  of  chemical  reaction  and  thinks 
that  the  problem  is  to  be  solved  in  accordance  with  the  laws  of 
physics.  As  we  have  seen  in  a  preceding  chapter,  Bordet  supposes 
that  both  in  agglutination  and  in  bacteriolysis  there  is  a  substance  in 
the  serum  which  renders  the  blood  and  bacterial  cells  sensitive  to  the 
action  of  a  ferment.  This  substance  he  calls  the  sensitizer,  and  he 
states  that  the  phenomenon  of  agglutination  is  similar  to  that  of  co- 
agulation, and  in  explaining  the  latter  he  endeavors  to  exclude  the 
necessity  of  the  presence  of  bodies  which  have  other  than  physical 
action. 


11 


CHAPTER   IX. 

IMMUNITY. 

It  is  a  matter  of  observation  as  old  as  the  history  of  man  that  all 
animals  are  not  alike  aifected  by  the  same  diseases,  and  that  epi- 
demics which  decimate  certain  species  are  wholly  without  effect  upon 
the  health  and  life  of  others.  Primitive  man  undoubtedly  recognized 
the  fact  that  one  attack  of  certain  diseases  gives  a  more  or  less  per- 
manent immunity  to  the  same  disorder.  It  has  also  been  long  recog- 
nized that  certain  diseases,  such  as  diphtheria  and  scarlet  fever,  which 
are  so  highly  fatal  among  children,  but  rarely  attack  adults.  The 
question  of  immunity  has  long  been,  is,  and  probably  will  long  con- 
tinue to  be  one  of  the  most  perplexing  problems  which  medical 
science  has  attempted  to  solve.  With  so  many  chances  of  infection, 
the  question  is  often  asked  how  is  it  that  the  human  race  continues 
its  existence,  and  why  has  it  not  been  swept  out  of  the  world  by 
epidemics  ?  The  answer  to  this  question  is  not  easy,  and  the  time 
has  not  come  when  it  can  be  given  with  perfect  satisfaction.  How- 
ever, we  are  now  in  possession  of  numerous  facts  bearing  on  this 
point,  and  it  will  be  our  attempt  in  this  chapter  to  briefly  state  the 
most  important  of  these. 

It  should  be  plainly  understood  that  the  factors  involved  in  secur- 
ing immunity  against  infectious  diseases  are  multiple  in  number  and 
varied  in  character.  A  mistake  has  been  made  in  endeavoring  to 
explain  immunity  as  being  due  to  one,  or  even  to  a  few  anti-bac- 
terial properties  of  the  animal  body.  This  has  been  admirably 
pointed  out  by  Meltzer,^  from  whom  we  make  the  following  quota- 
tion :  "  I  maintain  in  the  first  place  that  in  the  struggle  against 
bacteria  the  defense  of  the  body  is  not  carried  on  exclusively  or 
chiefly  by  a  single  element.  It  is  neither  the  body  fluids,  nor  the 
leucocytes,  nor  the  other  cells  alone  which  can  claim  the  exclusive 
merit  of  maintaining  the  health  of  the  body,  but  each  and  every  one 
of  them  has  its  variable  share  in  attaining  the  desired  end.  .  .  . 
Let  us  take  as  an  illustration  the  protection  of  the  conjunctival  sac. 
It  is  nearly  in  direct  contact  with  the  air,  and  we  might  expect  to 
find  there  an  extensive  bacterial  settlement.  Nevertheless  Lacho- 
witz  and  Bujwid  found  that  in  69  per  cent,  of  cases  the  conjunctiva 
was  perfectly  sterile.  The  factors  which  accomplish  this  sterility, 
or  at  least  comparative  sterility,  of  bacteria  are  :  The  .reflex  which 
causes  the  closure  of  the  lids  at  the  approach  of  dust  (the  carrier  of 

1  Congress  of  Am.  Physicians  and  Surgeons,  1900. 
162 


IMMUNITY.  163 

bacteria) ;  the  blinking  which  occurs  regularly  a  few  times  in  a  min- 
ute, which  in  conjunction  with  the  lachrymal  moisture,  throws  out 
again  mechanically  the  already  landed  bacteria  ;  and  finally,  the  bac- 
tericidal effect  of  the  tears  destroys  the  balance  of  the  invaders. 
Or  let  us  take  the  respiratory  organ  from  the  larynx  down  to  the 
respiratory  tissue,  including  the  corresponding  lymph  glands.  .  .  . 
Through  this  path  the  outside  world  stands  in  an  intimate  relation 
with  the  interior  of  the  body ;  inasmuch  as  the  air  column  is  sepa- 
rated from  the  lymphatics  and  capillaries  of  the  lungs  merely  by  a 
single  layer  of  the  very  thin  epithelium  of  the  air  cells.  Even  the 
serous  cavities  are  separated  from  the  lymph  spaces  by  thicker  lay- 
ers. This  arrangement  is,  of  course,  indispensable  for  the  proper 
exchange  of  the  blood  gases  with  the  air.  But  what  prevents  the 
bacterial  invasion  of  the  interior  of  the  body  by  this  open  and  direct 
way?  Moreover  most  of  the  writers  agree  that  trachea,  bronchi 
and  lung  tissue  of  healthy  animals  are  entirely  sterile.  In  a  num- 
ber of  rabbits  under  morphine  anesthesia  I  found  all  these  parts  to 
be  sterile.  If  one  vagus  or  a  laryngeal  branch  was  cut,  then  the  upper 
part  of  the  trachea  contained  bacteria,  but  not  the  lung.  When 
both  vagi  were  cut,  then  of  course  the  lungs  too  were  invaded. 
Jundell  reported  recently  that  by  means  of  a  special  device  he  was 
able  to  test  the  human  trachea,  and  found  that  in  the  majority  of 
healthy  cases  the  region  below  the  glottis  proved  to  be  sterile.  What 
protects  this  path  ?  In  my  opinion  the  result  is  accomplished  by  the 
cooperation  of  the  following  factors  :  The  tortuous  part  of  the  res- 
piratory path  lying  above  the  glottis  removes  perhaps  the  greatest 
part  of  the  bacteria  contained  in  the  inspired  air  column,  and  the 
remaining  number  is,  under  normal  conditions,  just  small  enough  to 
be  disposed  of  by  the  factors  present  below  the  larynx.  Bacteria 
which  pass  the  glottis  are  either  carried  back  outside  of  the  glottis 
from  the  trachea  and  the  bronchi  by  the  steady  movements  of  the 
cilia  of  the  epithelium,  or,  if  the  germs  are  carried  in  the  center  of 
the  air  column  down  to  the  air  cells,  they  quickly  penetrate  the  thin 
epithelial  layer  and  are  immediately  within  the  reach  of  the  lymph 
glands,  which  take  good  care  of  them.  ...  In  connection  with 
the  respiratory  path,  I  would  like  to  recall  here  the  interesting  fact 
that  both  canals  which  lead  farthest  to  the  innermost  of  the  body, 
that  is,  the  respiratory  and  the  female  genital  canal  (which  latter 
terminates  in  the  peritoneum),  have  ciliated  epithelium,  the  move- 
ments of  the  cilia  being  outward,  and  are,  as  far  as  the  epithelium 
extends,  entirely  or  nearly  sterile." 

Some  of  the  factors  which  lead  to  immunity  in  the  infectious  dis- 
eases pertain  to  the  animal  organism,  while  others  are  due  to  limita- 
tions of  the  capability  of  growth  on  the  part  of  the  infecting  agents. 
For  instance,  but  few  if  any  of  the  pathogenic  bacteria  are  able  to 
penetrate  the  unbroken  skin,  and  infection  through  this  avenue  can 


164  IMMUNITY. 

occur  only  when  there  is  some  break  in  the  continuity  of  the  cover- 
ing of  the  body.  Indeed  some  of  the  most  powerful  pathogenic 
microorganisms  are  practically  harmless  when  introduced  into  the 
body  subcutaneously,  while  they  may  speedily  cause  death  when 
taken  into  the  alimentary  canal.  This  is  true  of  the  vibrio  of  Asiatic 
cholera.  On  the  other  hand  tetanus  and  anthrax  bacilli  may  do  no 
harm  when  taken  into  a  healthy  stomach,  while  they  may  cause 
speedy  death  when  injected  subcutaneously.  These  examples  make 
it  evident  that  it  is  not  sufficient  for  a  pathogenic  microorganism, 
even  one  to  which  man  is  highly  susceptible,  to  come  in  contact  with 
the  human  body  in  order  to  cause  disease,  but  it  must  first  find  its 
suitable  port  of  entry,  and  even  after  this  has  been  reached,  there 
are  other  dangers  to  the  life  of  the  invader  with  which  he  may  have 
to  meet  before  he  reaches  the  place  where  he  can  entrench  himself 
and  begin  the  struggle.  Such  guards  are  furnished,  as  has  already 
been  seen,  by  the  mechanical  movements  of  epithelial  cilise,  by  the 
tortuosity  of  the  passage,  by  coming  in  contact  with  such  fluids  as 
the  gastric  juice,  or  by  being  hurried  through  the  body  and  cast  out 
in  the  excretions,  as  undoubtedly  sometimes  happens  to  typhoid 
bacilli  when  taken  into  the  alimentary  canal.  Moreover  every  toxi- 
cogenic  germ  is  not  capable  of  maintaining  even  for  a  short  time  a 
parasitic  existence.  The  obligate  saprophytes,  which  are  numerous, 
and  some  of  which  undoubtedly  produce  powerfiil  toxins,  are  capable 
of  harming  man  only  under  exceptional  conditions  which  are  spe- 
cially favorable  to  their  growth  and  multiplication.  Indeed,  some 
members  of  this  class  do  not  appear  to  be  capable  of  growth  and 
reproduction  when  introduced  into  any  part  of  the  animal  body,  and 
consequently  they  are  harmful  to  man  only  when  their  already  formed 
toxins  are  introduced  into  his  body.  These  bacteria  in  and  of  them- 
selves are  not  able  to  aflPect  the  health  or  endanger  the  life  of  man. 
Other  powerful  toxicogenic  bacteria  have  their  intracellular  toxins 
locked  up  in  cell  walls  which  under  ordinary  conditions  are  imper- 
meable ;  and  it  is  only  when  some  unusual  condition  gives  oppor- 
tunity for  the  disintegration  of  the  bacterial  cell  that  its  toxin  is 
liberated  and  may  do  harm.  This  seems  to  be  true  of  the  colon 
bacillus,  which  is  a  normal  habitant  of  the  intestines  of  man,  and 
which  contains  a  powerful  intracellular  poison.  Under  ordinary 
conditions  this  bacterium  is  harmless  to  man,  because  its  toxin  does 
not  diffuse  from  the  bacterial  cell,  and  consequently  cannot  be  ab- 
sorbed by  the  intestinal  wall ;  but  if  the  colon  bacillus  finds  its  way 
into  the  peritoneal  cavity,  where  its  cell  wall  is  disintegrated  by 
phagocytic  or  other  action,  its  toxin  is  liberated  to  the  great  danger 
of  the  host. 

Animals  differ  widely  not  only  in  their  susceptibility  to  bacterial  in- 
fection, but  also  to  bacterial  intoxication.  For  instance  certain  ani- 
mals, especially  birds  and  reptiles,  are  not  susceptible  to  the  toxin  of 


IMMUNITY.  165 

tetanus,  and  mice  and  rats  bear  large  doses  of  the  diphtheria  toxin 
■without  ill  eifect.  On  the  other  hand,  many  animals  that  are  sus- 
ceptible to  the  toxins  cannot  be  infected  with  the  bacteria  which  pro- 
duce these  toxins.  It  will  be  seen  from  this  that  there  may  be  both 
bacterial  and  toxin  immunity. 

We  may  discuss  the  subject  of  immunity  in  detail  under  the  fol- 
lowing heads  :  (1)  Natural  immunity,  or  that  possessed  by  certain 
species  or  races  of  animals  at  all  times  against  certain  diseases ;  (2) 
inherited  immunity,  which  may  be  transmitted  from  the  mother  to 
the  fetus  through  the  placental  circulation,  or  from  the  mother  to  the 
child  through  the  milk  ;  (3)  acquired  immunity,  or  that  which  is 
secured  by  one  attack  of  the  disease,  by  vaccination,  or  by  repeated 
treatments  with  sterilized  or  unsterilized  cultures.  Acquired  im- 
munity may  be  either  active  or  passive.  By  an  active  immunity  we 
mean  that  form  which  is  induced  by  the  direct  treatment  of  the  ani- 
mal with  filtered  or  unfiltered  cultures,  and  in  which  case  the  anti- 
toxic or  anti-bacterial  substance  is  produced  in  the  body  of  the  animal 
thus  treated.  Passive  immunity,  on  the  other  hand,  may  be  secured 
by  injecting  the  blood  serum  of  an  animal  actively  immunized  into  a 
second  animal.  As  an  illustration  of  active  and  passive  immunity,  we 
may  refer  to  the  method  by  which  diphtheria  antitoxin  is  prepared. 
The  horse  is  treated  with  successive,  non-fatal,  gradually  increased 
doses  of  diphtheria  toxin  until  this  animal  furnishes  a  serum  which 
contains  a  large  amount  of  diphtheria  antitoxin.  The  horse's  serum 
when  injected  into  the  child  sick  with  diphtheria  supplies  the  anti- 
toxin, which  combines  with  the  toxin,  and  thus  protects  the  tissues 
of  the  child  against  the  injury  that  would  otherwise  be  inflicted  on 
the  cellular  elements  by  the  noxious  agent.  In  this  illustration  an 
active  immunity  is  induced  in  the  horse,  and  as  a  result  of  the  es- 
tablishment of  this  condition  certain  cells  within  the  animal  are 
stimulated  to  a  form  of  activity  by  which  the  antitoxin  is  generated. 
In  other  words,'^the  horse  treated  with  successive  doses  of  the  diph- 
theria toxin  comes  to  possess  an  active  immunity.  The  horse's 
serum  containing  the  antitoxin  is  injected  into  the  child,  which  for 
the  time  being  becomes  physiologically  a  part  of  the  horse,  and  pos- 
sesses only  a  passive  immunity. 

We  will  now  proceed  to  discuss  in  more  detail  the  subject  of  nat- 
ural immunity.  It  is  a  fact  of  common  observation  as  well  as  of  ex- 
perimental demonstration  that  the  lower  animals  are  wholly  immune 
to  certain  bacterial  infections  to  which  man  is  markedly  susceptible. 
For  instance,  among  men  typhoid  fever  is  one  of  the  grave  diseases, 
causing  great  morbidity,  and  increasing  to  a  considerable  extent  the 
mortality  lists  ;  while  among  the  lower  animals  this  disease  does  not 
occur  naturally,  nor  has  anyone  as  yet  been  able  to  induce  it  by  in- 
oculation with  the  bacillus.  It  is  true  that  many  of  the  lower 
animals  are  susceptible  to  the  typhoid  toxin,  but  a  true  typhoidal  in- 


166  IMMUNITY. 

fection  with  the  bacterium  of  this  disease  has  not  been  established, 
although  frequently  tried,  in  any  of  the  animals  upon  which  experi- 
ments have  been  made.  Leprosy,  scarlet  fever,  yellow  fever  and 
measles  are  other  diseases  which  inflict  themselves  upon  mankind, 
but  to  which  the  lower  animals  are  apparently  insusceptible.  Some 
infections  are  prevalent  among  certain  species  of  animals,  while  upon 
others  they  seem  to  be  without  effect.  For  instance,  anthrax  is 
common  among  cattle  and  sheep,  while  many  carnivorous  animals 
wholly  escape  this  infection.  It  is  an  interesting  fact  that  in  na- 
ture some  animals,  such  as  the  guinea-pig  and  rabbit,  are  not  known 
to  suffer  from  epidemics  of  anthrax,  but  this  disease  can  be  easily  in- 
duced in  these  animals  by  artificial  inoculation.  Toxin  and  bacterial 
immunity  are  sometimes,  but  not  always,  possessed  by  the  same 
animal.  Rats  and  mice  are  insusceptible  to  both  the  bacillus  and 
the  toxin  of  diphtheria,  but,  as  we  have  already  seen,  many  animals 
succumb  to  the  typhoid  toxin,  while  none  are  susceptible  to  this 
infection. 

One  of  the  important  factors  in  natural  immunity  lies  in  the  fact 
that  the  toxicogenic  germ  is  unable  to  multiply  in  the  animal  body. 
The  bacillus  pyocyaneus  is  frequently  found  upon  the  surface  of 
man's  body,  especially  in  the  axillary  and  inguinal  regions,  and 
sometimes  it  occurs  in  the  intestines,  but  notwithstanding  this  al- 
most constant  proximity  of  this  organism  man  is  but  rarely  injured 
by  it,  yet,  nevertheless,  this  bacillus  produces  a  toxin  to  which  man 
is  susceptible.  The  micrococcus  prodigiosus  is  not  classed  among 
the  pathogenic  bacteria,  and  yet  experimentation  has  shown  that  its 
toxin,  which  has  been  used  in  the  treatment  of  malignant  growths, 
has  a  marked  effect  upon  man.  It  is  probable  that  most  of  the  sa- 
prophytic bacteria  contain  intracellular  substances  which  when  re- 
peatedly injected  into  animals  in  relatively  large  amounts  are  capable 
of  causing  death.  The  sarcines  are  usually  regarded  as  altogether 
harmless,  but  it  has  been  shown  that  the  intra-peritoneal  injection  of 
the  cellular  elements  of  these  organisms  is  followed  by  death.  It 
has  been  found  that  large  amounts  of  the  spores  of  the  tetanus  bacil- 
lus, when  completely  freed  from  the  toxin,  may  be  injected  into  rab- 
bits and  guinea-pigs  with  no  further  injury  than  the  formation  of  a 
pocket  of  pus  at  the  place  of  injection,  but  these  animals  are  highly 
susceptible  to  tetanus  toxin,  which  causes  in  them  typical  symptoms 
of  the  disease  and  induces  speedy  death.  The  natural  immunity 
possessed  by  some  animals  to  tetanus  infection  may  be  overcome  by 
the  employment  of  mixed  cultures,  even  when  the  accompanying 
microorganism  is  apparently  wholly  without  effect.  A  like  result 
may  be  secured  by  the  simultaneous  introduction  into  the  animal 
body  of  certain  chemicals,  such  as  lactic  acid.  On  the  other  hand, 
certain  animals  are  quite  insusceptible  to  the  toxin,  and  at  the  same 
time  easily  infected  with  the  microorganism.     A  healthy  man  is  rela- 


IMMUNITY.  167 

tively  insusceptible  to  tuberculin,  but  is  easily  invaded  by  the  tuber- 
cle bacillus,  and  after  becoming  infected  with  the  germ  he  loses  his 
insusceptibility  to  the  toxin.  It  may  be  seen  from  the  illustrations 
that  have  already  been  given  that  inability  on  the  part  of  the  micro- 
organisms to  grow  in  the  animal  body  is  an  important  factor  in  nat- 
ural immunity,  but  this  is  by  no  means  the  sole  factor  in  the  estab- 
lishment of  this  condition. 

In  the  chapter  on  the  germicidal  constituents  of  the  blood  we 
have  shown  the  existence  of  alexins  not  only  in  this  fluid  but  in 
other  tissues  and  juices  of  the  body.  It  was  at  one  time  supposed 
that  the  alexins  of  the  blood  played  a  large  part  in  the  establish- 
ment of  natural  immunity,  but  more  extended  investigation  has 
shown  that  this  supposition  is  erroneous.  The  blood  serum  of  the 
rabbit  has  relatively  strong  bactericidal  effect  upon  anthrax  bacilli, 
and  yet  this  animal  is  most  highly  susceptible  to  this  infection. 
Moreover,  Lubarsch  ^  has  shown  that  the  bactericidal  action  of  rab- 
bit's blood  on  the  anthrax  bacillus  is  more  marked  outside  than  it  is 
inside  the  body.  The  discovery  of  the  germicidal  effect  of  rat's 
blood  on  anthrax  bacilli  was  at  one  time  suggested  by  Behring,^  as 
an  explanation  of  the  relative  immunity  possessed  by  this  animal  to 
anthrax  infection.  However,  more  extended  observation  showed 
that  the  white  rat  is  more  susceptible  to  anthrax  than  was  at  one 
time  supposed,  and  yet  the  bactericidal  action  of  rat's  blood  on  the 
anthrax  bacillus  can  be  demonstrated  not  only  outside  the  animal 
body,  but  also  within  the  organism.  If  anthrax  bacilli  be  mixed 
with  rat's  blood  and  the  mixture  be  injected  into  mice,  the  latter 
animals  escape  infection,  but  the  rat  from  which  the  bactericidal 
blood  was  obtained  succumbs  to  infection  with  the  bacillus.  On  the 
other  hand,  the  adult  dog  is  immune  to  anthrax  infection,  while  the 
blood  serum  of  this  animal  has  no  germicidal  action  on  this  organ- 
ism. Indeed,  anthrax  bacilli  grow  abundantly  in  the  blood  serum 
of  the  dog.  It  will  thus  be  seen  that  the  blood  of  the  rabbit,  an 
animal  highly  susceptible  to  anthrax  infection,  has  marked  germicidal 
action  on  the  anthrax  bacilli  outside  the  body ;  while  the  blood  of 
the  dog,  an  animal  immune  to  anthrax  infection,  furnishes  a  good 
culture  medium  for  the  growth  of  the  bacillus.  We  must,  therefore, 
conclude  that  the  attempt  to  explain  natural  immunity  solely  by  the 
bactericidal  properties  of  the  blood  must  be  abandoned.  Indeed, 
it  is  probable  that  the  germicidal  properties  of  the  blood  do  not  con- 
stitute a  very  important  factor  in  the  production  of  natural  immunity. 
However,  we  do  not  claim  that  the  alexins  can  be  wholly  disre- 
garded in  the  study  of  this  problem,  and  after  bringing  forward 
another  theory  concerning  natural  immunity,  we  will  return  to  this 
subject. 

1  Centrcdblattf.  Bakteriologie,  6,  1889. 
^  Zeitschrift  fur  Hygiene,  9,  1890. 


168  IMMUNITY. 

According  to  Metschnikoff,  the  most  important  factor  in  the  pro- 
duction of  natural  immunity  is  to  be  found  in  the  phagocytic  action 
of  certain  cells  within  the  animal  body.  He  divides  phagocytes 
into  mobile  and  fixed  elements.  Among  the  former  he  places  both 
the  mono-  and  polynuclear  leucocytes  (with  the  exception  of  the 
small  lymphocytes,  and  the  mast  cells  of  Ehrlich),  and  the  so-called 
wandering  cells  ;  while  the  fixed  phagocytes,  or  macrophages,  consist 
of  endothelial  cells,  the  elements  of  the  spleen  pulp  and  of  bone 
marrow,  some  connective  tissue  cells,  and  possibly  certain  nerve  and 
muscle  cells.  He  states  that  sometimes  many  mononuclear  phago- 
cytes fuse  together  forming  large  plasmodia,  which  may  be  known 
as  giant  cells,  and  which  have  a  phagocytic  action.  When  bacteria 
are  introduced  into  a  naturally  immune  animal  they  are  seized  upon 
and  devoured  by  either  the  mobile  or  fixed  phagocytes,  or  by  both. 
When  a  microorganism  is  introduced  into  a  place  where  no  phago- 
cytes are  present,  as  for  instance  under  the  skin,  into  the  cornea,  or 
into  the  anterior  chamber  of  the  eye,  the  mobile  phagocytes  collect 
at  the  point  of  bacterial  invasion,  engulf  the  bacterial  cells  with  the 
aid  of  their  pseudopodia  and  then  digest  them  or  in  some  other  way 
deprive  them  of  their  capability  of  harming  the  body.  This 
method  of  disposing  of  foreign  substances  introduced  into  the  ani- 
mal body  is  known  as  phagocytosis.  Even  those  who  have  brought 
the  best  arguments  against  Metschnikoff 's  doctrine  of  phagocytosis 
admit  that  this  phenomenon  is  especially  observable  in  animals 
which  are  relatively  immune  to  the  invading  microorganism,  or  in 
other  words,  they  recognize  the  fact  that  in  natural  immunity  pha- 
gocytosis is  at  least  a  frequently  observed  phenomenon.  Pfeiffer 
and  Kolle,^  in  studying  the  former's  reaction,  which  has  already 
been  referred  to  in  our  chapter  on  lysins,  state  that  the  greater 
number  of  bacteria  destroyed  in  the  peritoneum  of  normal  ani- 
mals are  taken  up  by  leucocytes,  but  that  some  perish  in  the 
exudative  fluid.  Kruse  ^  states  that  phagocytosis  is  frequently  ob- 
served, especially  in  relatively  insusceptible  animals,  and  when  a 
weak  virus  is  used.  When  anthrax  bacilli  are  injected  under  the 
skin  of  a  dog  a  very  energetic  phagocytosis  takes  place  and  indeed, 
according  to  Denys  and  Havet,^  this  phenomenon  may  be  observed 
in  extra-vascular  blood  from  the  dog.  Even  spores  are  taken  up  by 
the  phagocytes  and  sometimes  they  develop  into  bacilli  within  the 
phagocyte,  thus  destroying  the  organism  which  has  engulfed  them, 
but  ordinarily  their  development  is  rendered  impossible,  and  their 
destruction  finally  accomplished.  The  phenomenon  of  phagocytosis 
can  be  explained  only  on  the  assumption  that  the  phagocyte  contains 
some  chemical  substance  by  virtue  of  which  it  destroys  the  captured 

^  Zeiischrifl  fiir  Hygiene,  21,  1896. 
'Flugg^s  Mikroorganismen,  1895. 
3ia  CeUiUe,  10,  1894. 


IMMUNITY.  169 

microorganism.  What  this  chemical  poison  is,  or  whether  it  is  the 
same  in  all  phagocytes,  we  cannot  at  present  determine.  All  phago- 
cytes contain  nucleic  acid,  and  the  germicidal  properties  of  this  have 
been  abundantly  demonstrated,  but  whether  or  not  there  may  be 
other  and  more  powerful  bactericidal  agents  in  certain  phagocytes,  we 
have  no  means  at  present  of  knowing.  It  is  still  a  question  whether 
or  not  the  germicidal  substance  contained  in  the  leucocytes  is  a  secre- 
tion of  these  organisms  or  a  result  of  their  disintegration.  As  has 
already  been  stated  in  the  chapter  on  the  germicidal  constituents  of 
the  blood,  it  seems  quite  evident  that  the  bactericidal  substances  in 
the  blood  serum  result  from  the  disintegration  of  the  white  corpuscles. 
It  is  possible  that  a  blood  which  furnishes  a  highly  bactericidal  serum 
gives  oif  but  little  or  no  germicidal  substance  to  the  plasma  in  the 
living  body.  It  is  probable  that  the  leucocytes  in  the  blood  of 
certain  animals  disintegrate  more  rapidly  when  removed  from  the 
body  than  happens  to  the  leucocytes  of  other  animals.  In  instances 
of  the  first  kind  we  would  expect  to  find  the  blood  serum  possessed 
of  marked  germicidal  properties,  while  in  cases  of  the  second  kind 
the  serum  may  be  quite  devoid  of  bactericidal  action.  Again  it  is 
possible  that  the  phagocytes  in  the  blood  of  one  animal  may  retain 
their  germicidal  constituents  longer  than  do  the  leucocytes  in  the 
blood  of  other  animals.  In  the  serum  of  a  blood  of  the  first  kind  we 
would  not  expect  to  find  a  large  amount  of  germicidal  substance  and 
yet  within  the  body  the  phagocytes  of  this  animal  may  be  more 
eflPective  in  the  destruction  of  germs  than  are  those  in  the  blood  of 
an  animal  whose  serum  is  rich  in  germicidal  substances. 

There  is  another  chemical  process  in  the  phenomenon  of  phago- 
cytosis which  is  of  the  greatest  importance.  The  phagocytes  are 
attracted  by  the  introduction  into  the  body  of  certain  substances  and 
repelled  by  others.  This  is  known  as  chemotaxis,  which  may  be 
either  positive  or  negative.  In  other  words,  the  foreign  substance 
introduced  into  the  animal  body  may  attract  the  phagocytes,  or  it 
may  repel  them.  In  the  former  instance  the  phagocytes  will  gather 
in  large  numbers  about  the  foreign  substance  introduced  into  the 
body,  and  phagocytic  action  will  be  most  marked.  It  appears  to  us 
that  the  greatest  factor  in  natural  immunity,  so  far  as  the  action  of 
the  blood  of  the  body  on  the  invading  microdrganism  is  concerned, 
lies  in  the  fact  that  in  natural  immunity  chemotaxis  is  positive,  and 
the  more  marked  the  natural  immunity  is,  the  stronger  is  the  posi- 
tive chemotaxis.  It  should  be  understood  that  at  present  we  have 
reference  only  to  natural  immunity  against  infection,  and  that  we  do 
not  include  in  these  statements  our  opinions  concerning  natural  im- 
munity against  toxins. 

That  bacteria  may  retain  their  vitality  after  being  taken  into  the 
phagocyte  has  been  demonstrated  by  injecting  phagocytes  containing 
bacilli  into  susceptible  animals  and  thus  inducing  infection.     When 


170  IMMUNITY. 

phagocytes  containing  bacteria  are  removed  from  the  body  the  phago- 
cytes die  and  some  of  the  contained  bacteria  demonstrate  their  re- 
tention of  life  by  growth  and  multiplication.  It  sometimes  happens 
within  the  body  that  the  bacterium  proves  too  powerful  an  opponent, 
and  the  phagocyte  is  destroyed.  This  condition  can  be  brought 
about  by  the  employment  of  a  highly  virulent  culture  or  by  placing 
the  animal  after  inoculation  under  unfavorable  conditions.  For  in- 
stance Pasteur  and  Joubert  demonstrated  that  while  the  chicken  is 
naturally  immune  to  anthrax,  susceptibility  may  be  secured  by  keep- 
ing the  inoculated  animal  at  a  low  temperature,  under  the  unfavor- 
able influence  of  which  normal  phagocytic  action  is  not  possible. 

It  is  probable  that  plasmolysis  is  also  a  factor  in  natural  immu- 
nity. It  is  a  well-known  fact  that  the  removal  of  bacteria  from  one 
medium  into  another  containing  slightly  more  or  less  of  certain  min- 
eral constituents  may  be  followed  by  the  death  of  all  or  the  greater 
part  of  the  microorganisms.  Bacteria  consist  of  cell-walls  with  con-, 
tents.  Diffusion  through  the  cell-wall  is  constantly  taking  place  to 
a  greater  or  less  extent.  By  altering  the  mineral  constituents  of  the 
medium,  an  osmosis,  which  may  prove  fatal  to  the  bacterium,  results. 
The  removal  of  the  bacterial  cell  from  an  isotonic  medium  to  one 
which  is  either  hypotonic  or  hypertonic  leads  to  greater  or  less  injury 
to  the  cell. 

The  subject  of  natural  immunity  to  toxins  is  one  of  quite  as  much 
interest  as  that  which  we  have  just  been  discussing.  Instances  of 
insusceptibility  to  powerful  poisons  have  long  been  known  and  have 
formed  the  subject-matter  of  much  theoretical  discussion.  For  in- 
stance, hogs  can  eat  with  impunity  relatively  large  amounts  of  arsenic 
and  antimony,  and  certain  insects  not  only  live,  but  seem  to  thrive 
in  an  atmosphere  saturated  with  the  vapor  of  formaldehyde.  Metsch- 
nikoff  states  that  there  are  certain  invertebrates  which  are  not  af- 
fected by  large  doses  of  some  of  the  most  important  bacterial  toxins. 
The  larva  of  a  large  beetle  (Nashornkafer)  has  been  found  to  be 
wholly  immune  to  the  cholera  toxin.  If  one  of  these  and  a  small 
green  frog  of  equal  weight  be  treated  with  0.5  c.c.  of  soluble  cholera 
toxin,  the  larval  beetle  remains  apparently  unaffected,  while  the  frog 
speedily  dies.  On  the  other  hand,  if  both  animals  be  inoculated  with 
the  cholera  bacillus,  the  beetle  succumbs  to  cholera  sepsis,  while  the 
frog  escapes  infection.  Metschnikoff  explains  these  observed  facts 
on  the  ground  that  the  leucocytes  of  the  frog  consume  the  vibrios, 
but  are  powerless  against  the  toxin,  while  the  leucocytes  of  the  lar- 
val beetle  are  unable  to  cope  with  the  bacteria,  but  it  must  be  ad- 
mitted that  he  does  not  explain  how  it  is  that  the  beetle  succumbs  to 
the  cholera  toxin  elaborated  in  its  own  body,  while  it  is  insusceptible 
to  that  introduced  from  without. 

It  is  well  known  that  certain  vertebrates  are  very  resistant  to  the 
most  potent  venoms  of  snakes.     This  is  notably  true  of  the  mon- 


IMMUNITY.  171 

goose  and  hedgehog,  and  less  markedly  so  of  hogs.  The  crocodile 
is  insusceptible  to  both  the  tetanus  bacillus  and  its  toxin,  but  is  sus- 
ceptible to  diphtheria  toxin.  Many  birds,  including  the  barnyard 
fowl,  are  insusceptible  to  the  tetanus  toxin.  The  best  explanation 
that  has  been  given  to  natural  immunity  against  toxins  is  that  offered 
by  Ehrlich's  theory.  This  author  holds  that  in  order  for  a  substance 
to  be  toxic  to  the  animal  organism  the  latter  must  contain  cells,  the 
molecules  of  which  furnish  side  chains  capable  of  forming  chemical 
compounds  with  the  toxin.  When  the  toxin  is  not  capable  of  form- 
ing a  chemical  combination  with  some  constituent  of  the  cell,  it  has 
no  poisonous  action  on  that  animal.  Some  bacterial  toxins,  such  as 
that  of  tetanus,  form  a  destructive  combination  with  the  cells  of  the 
central  nervous  system,  while  others  .possibly  form  similar  compounds 
with  other  tissues  of  the  body.  Therefore,  according  to  Ehrlich, 
natural  immunity  to  toxins  is  due  to  the  failure  to  form  combinations 
in  the  animal  body  which  are  destructive  to  some  of  its  cellular 
elements. 

The  study  of  acquired  immunity  has  furnished  a  rich  field  for 
research  to  the  bacteriologist.  Early  in  his  development  man  must 
have  observed  that  one  attack  of  certain  diseases  gave  to  those  who 
recovered  more  or  less  permanent  immunity  to  that  disease.  The 
Chinese  recognized  this  fact  many  centuries  ago,  so  far  as  smallpox 
is  concerned,  and  this  led  to  their  practice  of  inoculation  for  this 
disease.  This  custom  probably  spread  from  the  Orient  through 
Tartar  tribes,  and,  as  is  well  known,  was  introduced  into  England 
from  Turkey.  The  next  step  that  was  made  in  the  study  of  acquired 
immunity  was  the  discovery  of  vaccination  for  smallpox  as  practiced 
by  Jenner ;  but  inasmuch  as  up  to  that  time  the  specific  bacteria 
remained  unknown,  the  philosophy  of  vaccination  could  not  be  ex- 
plained. The  scientific  study  of  acquired  immunity  may  be  said  to 
date  from  the  early  investigations  of  Pasteur,  who  in  1880  discov- 
ered that  inoculations  with  non-virulent  cultures  of  chicken  cholera 
gave  immunity  to  subsequent  inoculations  with  a  virulent  culture 
of  this  microorganism.  Indeed,  as  early  as  1877  Pasteur  observed 
that  susceptible  animals  inoculated  with  cultures  of  the  anthrax 
bacillus  mixed  with  other  bacteria  did  not  acquire  the  disease.  This 
discovery,  as  we  shall  see  later,  has  been  amplified  by  others,  espe- 
cially by  Emmerich.  It  may  be  well  for  us  to  proceed  in  the  discus- 
sion of  acquired  immunity  by  considering  the  different  agents  em- 
ployed in  securing  this  condition.  They  are  as  follows  :  (1)  By 
treatment  with  weakened  cultures  of  the  germ  of  the  disease.  (2) 
By  the  employment  of  sterilized  cultures  of  the  specific  microorgan- 
ism or  by  the  use  of  its  toxins.  (3)  By  treating  the  animal  with 
cultures  of  the  specific  organism  mixed  with  other  bacteria,  or  by 
the  employment  of  mixed  cultures  in  which  the  specific  microorgan- 
ism is  present. 


172  IMMUNITY. 

Pasteur  ascertained  that  when  the  bacillus  of  chicken  cholera  is 
grown  on  artificial  culture  media  through  successive  generations,  it 
gradually  loses  its  virulence  and  finally  reaches  a  point  when  it  is  no 
longer  capable  of  inducing  the  disease  in  susceptible  animals.  Then 
he  found  that  animals  treated  with  this  non-virulent  culture  and  sub- 
sequently inoculated  with  virulent  cultures  remain  unaffected.  With 
the  anthrax  bacillus  it  was  found  that  there  is  no  loss  of  virulence  in 
successive  growths  on  artificial  media.  It  was  therefore  necessary 
to  resort  to  other  means  of  robbing  this  organism  of  its  virulence. 
Pasteur  found  that  this  could  be  accomplished  in  several  ways,  the 
most  important  of  which  were  :  (1)  Growth  at  relatively  high  tem- 
peratures, and  (2)  growth  in  culture  media  containing  small  amounts 
of  certain  antiseptics,  such  as  carbolic  acid.  By  both  of  these  meth- 
ods he  was  able  to  secure  vaccines  which  were  found  to  give  tempo- 
rary immunity.  Vaccines  of  this  kind  are  still  employed,  notably 
for  anthrax,  but  the  immunity  secured  in  this  way  is,  in  most  in- 
stances at  least,  of  relatively  short  duration,  and  this  method  cannot 
be  said  to  be  altogether  successful.  It  is  true  that  herds  of  cattle 
and  sheep  may  be  protected  from  a  prevailing  epidemic  in  this  way, 
but  revaccination  must  be  frequently  practiced,  and  there  is  always 
danger,  unless  great  care  is  exercised,  of  not  sufficiently  reducing  the 
virulence  of  the  germ,  and  epidemics  have  been  extended  by  the 
employment  of  imperfectly  prepared  vaccines. 

Attempts  to  secure  immunity  by  the  employment  of  sterilized 
cultures  were  probably  suggested  by  the  discovery  of  the  fact  that 
immunity  to  the  venom  of  snakes  may  be  obtained  by  frequent  suc- 
cessive injections  of  non-fatal  doses.  As  early  as  1886,  Sewall  im- 
munized pigeons  to  the  venom  of  the  rattlesnake,  and  soon  thereafter 
Salmon  and  Smith  immunized  animals  to  the  bacillus  of  hog  cholera 
by  successive  treatments  with  sterilized  cultures  of  the  bacillus  of 
this  disease.  The  later  researches  of  Ehrlich  with  abrin  and  ricin 
gave  great  impetus  to  study  along  these  lines,  and  space  cannot  be 
given  here  to  even  an  enumeration  of  all  the  work  that  has  been 
done  in  the  study  of  immunity  by  treatment  with  sterilized  cultures. 
In  some  instances  the  action  of  the  toxin  has  been  modified  by  heat- 
ing or  by  the  addition  of  certain  chemicals,  such  as  iodin  and  chlorin. 
The  production  of  immunity  by  the  employment  of  sterilized  cul- 
tures or  toxins  is  due  in  most  instances,  if  not  in  all,  to  the  formation 
of  antitoxins,  and  the  explanation  of  the  formation  of  these  bodies 
will  be  discussed  later. 

A  certain  degree  of  immunity  to  some  of  the  infectious  diseases 
may  be  secured  by  previous  treatment  of  the  animal  with  substances 
of  non-bacterial  origin.  The  first  hint  that  this  might  be  accom- 
plished was  offered  by  Wooldridge,  who  reported  that  he  had  secured 
immunity  against  anthrax  by  previous  treatment  of  the  animals  with 
extracts  of  testicles  and  thymus  gland.     Although  subsequent  inves- 


IMMUNITY.  173 

ligations  have  not  fully  confirmed  this  statement,  they  have  led  to 
the  discovery  of  many  interesting  facts.  It  has  been  found  that  at 
least  temporary  immunity  against  some  of  the  infections  may  be 
secured  by  previous  treatment  of  the  animal  with  normal  serum, 
nucleinic  acid,  bouillon,  or  even  physiological  salt  solution.  For 
instance,  Issaeflf,^  found  that  animals  can  be  protected  against  experi- 
mental cholera  peritonitis  by  previous  intra-peritoneal  injections  of 
the  above-mentioned  substances.  This  investigator  found  that  im- 
mediately after  the  preventive  injection  there  is  a  marked  diminution 
in  the  number  of  leucocytes  in  the  peritoneal  lymph,  but  after  a 
short  period  of  time  the  leucocytes  return  in  increased  numbers. 
Now  if  at  this  stage  the  ordinarily  fatal  quantity  of  a  culture  of  the 
cholera  vibrio  be  injected  into  the  peritoneum,  death  does  not  follow. 
In  accordance  with  the  phagocytic  doctrine  of  Metschnikoff  it  is  sup- 
posed that  these  substances  of  non-bacterial  origin  give  temporary 
immunity  on  account  of  the  phagocytosis  which  they  induce.  Tem- 
porary immunity  against  some  of  the  most  highly  virulent  bacteria 
may  be  secured  in  this  manner.  Thus  Bordet  succeeded  in  immuniz- 
ing animals  against  most  virulent  cultures  of  the  streptococcus  by 
previous  treatment  with  ordinary  nutritive  bouillon.  This  form  of 
securing  immunity  is  more  easily  demonstrated  by  peritoneal  inocu- 
lations than  in  any  other  way.  It  is  possible  that  the  injection  of 
the  preventive  substance  leads  to  more  or  less  phagolysis,  and  as  a 
result  of  this  the  peritoneal  fluid  becomes  unusually  rich  in  bacteri- 
cidal substances,  which  result  from  the  disintegration  of  the  phago- 
cytes. Metschnikoff  further  suggests  that  the  phagolysis  caused  by 
the  introduction  of  the  preventive  solution  is  the  active  agent  in 
causing  the  accumulation  of  phagocytes  at  the  place  where  the 
phagolysis  has  occurred,  which  in  this  instance  is  in  the  peritoneal 
cavity.  On  account  of  the  phagolysis,  due  to  the  introduction  of 
such  a  substance  as  bouillon,  the  fluid  content  of  the  peritoneal  cav- 
ity becomes  rich  in  dissolved  bactericidal  substances  and  in  this  re- 
spect resembles  certain  blood  sera.  When  the  bacteria  are  subse- 
quently injected  into  the  peritoneal  cavity  many  of  them  are  destroyed 
by  the  soluble  germicidal  substances,  and  those  that  escape  this 
action  are  seized  upon  by  the  incoming  phagocytes.  If  a  drop  of 
the  peritoneal  fluid  be  examined  at  this  time  it  will  be  observed  that 
some  of  the  bacteria  have  met  with  extra-cellular  death,  while  oth- 
ers are  engulfed  in  the  phagocytes. 

The  most  probable  explanation  of  the  good  results  obtained  in 
Pasteur's  treatment  of  hydrophobia  is  that  by  this  means  a  toxin 
immunity  is  secured.  The  organism  which  is  supposed  to  cause 
hydrophobia  has  not  as  yet  been  discovered,  but  the  probabilities 
are  that  inoculation  with  a  living  organism  is  made  by  the  bite  of 
the  rabid  dog.  In  this  disease  a  long  period  of  incubation  precedes 
^  Zeitschriftfur  Hygiene,  16,  1894. 


174  IMMUNITY. 

the  development  of  the  first  symptoms.  Soon  after  the  man  has 
been  bitten  he  is,  according  to  the  Pasteur  method,  treated  succes- 
sively with  dried  portions  of  spinal  cords  taken  from  animals  which 
have  died  from  experimental  hydrophobia.  These  cords  probably 
contain  the  toxin  of  the  disease,  or  the  modified  microorganism,  or 
both,  and  those  which  have  been  dried  longest  contain  these  in  their 
least  active  form.  By  successive  inoculations  with  emulsions  of 
these  cords  immunity  to  the  hydrophobia  virus  may  be  secured  be- 
fore the  period  necessary  for  the  development  of  the  germ  introduced 
with  the  bite  of  the  dog  has  passed. 

For  some  years  Emmerich  has  worked  on  a  theory  of  immunity, 
which,  if  it  should  prove  to  be  correct,  gives  a  simple  explanation  of 
many  phenomena  which  have  puzzled  bacteriologists.  Recently  this 
theory  has  been  elaborated  in  two  papers  published  by  Emmerich  and 
Low,^  and  its  important  points  may  be  stated  as  follows  :  Many 
bacteria,  and  among  these  some  of  the  most  important  pathogenic 
ones,  produce,  both  in  vitro  and  in  vivo,  enzymes  which  are  capable 
of  digesting  the  organism  which  produces  them,  and  sometimes  other 
bacteria  as  well.  In  proof  of  this  statement  they  bring  forward  the 
following  observation  :  If  a  culture  of  the  bacillus  pyocyaneus  be 
allowed  to  stand  at  37°  there  forms  after  three  days  on  the  surface  a 
thick  scum  consisting  of  bacilli.  If  the  tube  be  thoroughly  shaken 
some  of  the  bacteria  subside,  and  after  three  days  longer  a  new  scum 
forms  on  the  surface.  The  process  of  shaking  and  allowing  to  grow 
is  repeated  six  or  eight  times,  and  finally  it  will  be  observed  that 
only  a  very  small  residue  of  the  bacteria  remains  and  is  deposited  on 
the  bottom.  If  such  a  deposit  found  in  a  culture  a  few  weeks  old  be 
examined  microscopically  there  will  be  found  a  few  whole  bacilli 
which  still  stain  well  with  fuchsin,  but  the  mass  of  the  deposit  will 
be  seen  to  consist  of  broken-down  bacilli  which  do  not  stain  readily, 
drops  of  fat,  and  crystals.  They  explain  this  phenomenon  on  the 
ground  that  the  bacteria  have  produced  an  enzyme  which  finally 
manifests  its  bacteriolytic  action  on  the  germs.  If  this  experiment 
be  repeated  with  a  culture  of  the  bacillus  of  swine  erysipelas,  similar 
changes  will  be  observed.  The  first  eifect  of  the  enzyme  is  to  agglu- 
tinate the  bacilli,  while  the  final  result  is  a  bacteriolytic  one.  It 
will  be  seen  from  this  that  both  agglutination  and  bacteriolysis  are 
supposed  to  be  due  to  an  enzyme  produced  by  the  microorganism. 
The  difference  between  the  change  observed  in  artificial  cultures  and 
in  immune  serum  is  due  to  the  fact  that  the  latter  contains  a  larger 
quantity  of  ready-formed  enzyme,  while  in  the  former  the  enzyme  is 
gradually  produced.  Illustrations  of  similar  phenomena  are  drawn 
from  the  study  of  the  action  of  enzymes  on  the  cellulose  of  moulds. 
For  instance  certain  parasitic  moulds  pierce  the  wood  of  living  trees 
by  means  of  enzymes  produced  in  the  innermost  growths  of  the  mould, 
1  Zeitschr.  /.  Hygiene,  31,  1899  ;  36,  1901. 


IMMUNITY.  175 

and  by  which  the  cellulose  of  the  tree  is  digested.  After  a  time  these 
ferments  digest  not  only  the  cellulose  of  the  tree  but  also  the  older 
parts  of  the  mould  itself.  The  action  of  bacterial  enzymes  of  this 
class  is  oifered  as  an  explanation  of  the  degeneration  forms  observed 
in  many  old  bacterial  cultures.  From  such  cultures  there  may  be 
precipitated  with  alcohol  an  albuminous  body  which  when  dissolved 
in  feebly  alkaline  solution  has  a  bacteriolytic  effect.  Arrest  of 
growth  in  artificial  cultures  occurs  when  the  solution  of  the  bacteri- 
olytic enzyme  is  sufficient  to  digest  newly  formed  bacteria.  The 
same  phenomenon  is  offered  as  an  explanation  of  the  self-limitation 
of  the  infectious  diseases.  When  the  bacteriolytic  enzyme  formed  in 
the  animal  body  has  reached  a  sufficient  degree  of  concentration  to 
dissolve  the  bacteria  which  have  produced  it,  further  growth  of  the 
organism  is  impossible,  and  the  disease  is  arrested.  It  is  probable 
that  the  enzyme  exists  in  the  bacterial  cell  as  a  zymogen,  and  is  able 
to  manifest  its  digestive  action  only  after  liberation  from  the  cell 
contents.  The  enzyme  produced  by  a  given  bacillus  may  dissolve 
the  cell  membrane  not  only  of  the  bacterium  which  has  produced  it, 
but  of  other  bacteria  as  well.  These  enzymes  are  divided  into  two 
classes,  which  are  known  as  conforme  and  heteroforme,  the  former 
being  one  which  dissolves  only  the  bacterium  which  produced  it,  while 
the  latter  is  one  which  has  a  bacteriolytic  action  upon  other  micro- 
organisms as  well.  It  is  proposed  that  bacteriolytic  enzymes  be 
given  the  general  name  of  nucleases,  because  they  digest  the  nucleo- 
proteids  of  the  bacterial  cells.  Special  enzymes  are  designated  by 
names  derived  from  those  of  the  bacilli  which  produce  them.  Thus 
the  bacteriolytic  enzyme  of  the  bacillus  pyocyaneus  is  known  as  pyo- 
cyanase  ;  that  of  the  cholera  vibrio  as  cholerase,  etc.  It  is  supposed 
that  in  the  living  body  the  enzymes  of  pathogenic  bacteria  combine 
with  certain  albuminous  bodies,  probably  those  derived  from  the  leu- 
cocytes. The  substances  resulting  from  these  combinations  are  desig- 
nated immuneproteids,  and  one  of  these  is  distinguished  from  the 
others  by  prefixing  the  name  of  the  special  enzyme.  Thus  we 
have  pyocyanase-immuneproteid,  cholerase-immuneproteid,  typhase- 
immuneproteid,  etc. 

Emmerich  and  Low  have  shown  experimentally  that  pyocyanase 
readily  destroys  and  dissolves  anthrax  bacilli  in  vitro,  both  under 
aerobic  and  anaerobic  conditions.  They  have  also  shown  that  rab- 
bits inoculated  with  virulent  cultures  of  anthrax  do  not  die  if  they 
be  treated  with  solutions  of  pyocyanase.  In  these  experiments  the 
animals  received  their  first  treatment  simultaneously  with  the  inocu- 
lation or  directly  afterward,  and  subsequent  treatments  were  also 
given.  They  were  not  able  to  immunize  rabbits  to  anthrax  by  pre- 
vious treatment  with  solutions  of  pyocyanase.  However,  they  did 
succeed  in  preparing  artificially  pyocyanase-immuneproteids  with 
which  immunity  to  anthrax  was  secured.     In  these  experiments  con- 


176  IMMUNITY. 

centrated  solutions  of  pyocyanase  were  obtained  either  by  evapora- 
tion of  old  filtered  cultures  of  the  bacillus  pyocyaneus  in  vacuo,  or 
by  precipitation  of  the  enzyme  and  subsequent  solution  of  the  pre- 
cipitate. It  should  be  stated  that  Bouchard  has  successfully  treated 
anthrax  by  injection  of  cultures  of  the  bacillus  pyocyaneus,  and  also 
by  treatment  with  blood  serum  of  sheep  which  had  been  immunized 
to  the  streptococcus  of  erysipelas,  but  inasmuch  as  cultures  of  the  ba- 
cillus pyocyaneus  are  themselves  toxic,  Pettenkofer  very  properly  said 
of  this  treatment :  "  One  drives  out  the  devil  with  Beelzebub."  Em- 
merich and  Low  hold  that  the  pyocyanase  as  they  have  obtained  it 
is  free  from  toxic  action.  They  think  it  probable  that  the  poisonous 
constituents  of  filtered  cultures  of  this  bacillus  are  given  off  during 
the  process  of  evaporation  in  vacuo  or  are  separated  from  the  enzyme 
when  the  latter  is  precipitated  from  old  cultures.  At  least  it  ap- 
pears that  the  animals  which  they  treated  with  solutions  of  pyocya- 
nase were  not  injuriously  affected  by  the  injections.  Pyocyanase  also 
digests  typhoid,  diphtheria,  pest  and  cholera  bacilli. 

Pyocyanase,  as  precipitated  by  alcohol,  forms  a  yellowish-green 
amorphous  substance  which  is  readily  soluble  in  water,  to  which  it 
imparts  a  greenish  tint  similar  to  that  possessed  by  cultures  of  the 
bacillus.  The  aqueous  solution  is  distinctly  alkaline.  Both  Mil- 
Ion's  reagent  and  the  biuret  reaction  fail  when  applied  to  aqueous 
solutions  of  pyocyanase,  while  a  violet  coloration  is  produced  on 
heating  with  concentrated  hydrochloric  acid.  When  pyocyanase  is 
heated  with  caustic  potash  it  becomes  intensely  yellow,  and  on  the 
addition  of  lead  acetate  a  blackish  deposit  is  formed,  thus  indicating 
that  this  substance  contains  sulphur.  It  is  an  interesting  fact  that 
this  enzyme  may  be  heated  to  90°  without  loss  to  its  bacteriolytic 
activity,  and  a  temperature  of  98.5°  reduces,  but  does  not  wholly 
rob  it  of  this  property.  Notwithstanding  its  great  resistance  to  heat, 
Emmerich  and  Low  conclude  that  this  substance  must  be  classed 
among  the  enzymes  on  account  of  its  energetic  action  in  very  small 
amounts.  They  also  refer  to  the  investigation  of  Wiirtz,  who  has 
shown  that  the  ferment  papayotin  retains  its  peptonizing  action  after 
having  been  exposed  to  a  temperature  of  105°.  If  a  small  amount 
of  dry  pyocyanase  be  placed  in  a  tube  of  gelatin  and  be  kept  at  22° 
the  contents  of  the  tube  are  completely  peptonized  within  twenty- 
four  hours.     It  also  acts  energetically  upon  fibrin. 

Emmerich  and  Low  conclude  their  first  paper  with  the  following 
statements  :  (1)  The  fact  that  liquid  cultures  of  most  bacteria  cease 
to  develop  after  a  few  days  or  weeks  is  due  to  the  elaboration  by  the 
bacteria  of  enzymes,  which  after  reaching  a  certain  degree  of  concen- 
tration, dissolve  the  bacterial  cells.  (2)  There  are  bacteriolytic  en- 
zymes which  digest  not  only  their  own  bacteria,  but  others  as  well. 
(3)  The  curative  action  of  both  filtered  and  unfiltered  cultures  is  due 
to  the  presence  in  these  cultures  of  bacteriolytic  enzymes.     (4)  The 


IMMUNITY.  177 

production  of  artificial^immunity  with  filtered  or  unfiltered  cultures  of 
pathogenic  bacteria  is  due  to  the  gradual  formation  within  the  body 
of  a  compound  of  the  bacteriolytic  enzyme  with  some  albuminous 
body ;  the  resulting  immune  proteid  retains  the  bacteriolytic  activ- 
ity of  the  original  enzyme.  (5)  This  combination,  which  takes  place 
very  slowly  in  the  animal  body,  may  be  speedily  brought  about  by 
chemical  reagents  in  vitro.  (6)  So-called  agglutination  is  nothing 
more  than  the  first  stage  of  bacteriolytic  action  of  enzymes.  (7) 
Immune  sera  manifest  their  bactericidal  action  more  energetically 
under  anaerobic  than  under  aerobic  conditions,  but  even  in  the  pres- 
ence of  air,  the  bacterial  enzymes  when  in  highly  concentrated  solu- 
tion show  marked  bactericidal  action.  (8)  There  are  bacterial  en- 
zymes which  in  the  animal  body  destroy  not  only  the  bacterial  cells, 
but  their  toxins  as  well.  For  instance,  pyocyanase  protects  animals 
which  have  been  treated  with  lethal  doses  of  diphtheria  toxin.  (9) 
Our  experiments  show  that  pyocyanase  may  be  used  successfully  in 
the  treatment  of  anthrax,  diphtheria,  pest,  etc.  (10)  By  means  of 
artificially  prepared  pyocyanasorimmuneproteid  a  high  degree  of  im- 
munity against  anthrax,  lasting  for  at  least  fourteen  days,  may  be 
secured.  (11)  A  slight  elevation  of  temperature  (about  one  degree) 
observed  in  animals  with  anthrax  treated  with  pyocyanase  is  a  result 
of  the  injection  of  the  enzyme.  (12)  The  bactericidal  action  of 
normal  blood  is  probably  due  to  the  presence  of  enzymes. 

In  their  second  paper  Emmerich  and  L5w  detail  the  methods 
which  they  have  employed  in  the  preparation  of  their  nucleases  and 
immuneproteids.  The  organism  whose  enzyme  is  desired  is  grown 
in  a  medium  which  is  as  free  as  possible  from  albuminous  constituents. 
This  is  necessary  in  order  that  the  enzyme  employed  may  be  obtained 
on  precipitation  with  alcohol  without  mixture  with  foreign  albumi- 
nous substances.  As  a  culture  medium  for  the  bacillus  pyocyaneus, 
the  following  preparation  is  used  : 

Distilled  water,  1,000.0 

Asparagin,  5.0 

Sodium  acetate,  5.0 

Dipotassium  phosphate,  2.0 

Magnesium  sulphate,  0.1 

Sodium  chlorid,  2.0 

In  order  to  prepare  the  nucleases  in  large  quantities,  from  ten  to 
twelve  liters  of  the  above  medium  is  divided  in  portions  of  one-half 
liter  each  in  liter  flasks.  These  are  inoculated  and  allowed  to  grow  for 
some  weeks  at  25°,  and  then  for  some  weeks  longer  at  from  30°  to  37°. 
After  five  or  six  weeks  the  cultures  are  shaken  thoroughly  at  least 
once  in  twenty-four  hours,  and  when  finally  there  remains  only  a 
slight  deposit  of  germ  detritus,  the  supernatant  fluid  is  drained  oflf 
12 


178  IMMUNITY. 

by  means  of  a  vacuum  pump.  This  fluid  is  filtered  through  porce- 
lain and  evaporated  in  vacuo  at  from  20°  to  36°  to  one-tenth  its 
volume  or  less.  The  concentrated  enzyme  solution  is  deprived  of 
inorganic  salts  and  a  part  of  its  toxic  constituents  by  dialysis,  con- 
tinued from  twelve  to  twenty-four  hours.  If  after  this  treatment  the 
presence  of  toxic  substances  can  be  shown  by  animal  experimenta- 
tion, three-tenths  per  cent,  of  trikresol  is  added  and  the  solution 
allowed  to  stand  for  a  few  weeks  longer,  during  which  time  appar- 
ently the  bacteriolytic  enzyme  destroys  any  trace  of  toxic  substance 
that  may  be  present.  The  concentrated  solution  of  enzyme  thus  ob- 
tained has,  according  to  Emmerich  and  Low,  not  only  bactericidal 
properties,  but  also  the  capability  of  destroying  certain  toxins,  notably 
that  of  diphtheria,  in  the  animal  body.  As  has  been  stated,  these 
authors  were  not  able  to  immunize  animals  to  anthrax  with  the 
enzyme  of  the  bacillus  pyocyaneus,  but  this  object  they  were  able  to 
accomplish  by  the  employment  of  their  pyocyanase-immuneproteid. 
This  compound  they  have  prepared  in  several  ways,  one  of  which  is 
as  follows  :  To  each  100  c.c.  of  the  concentrated  and  dialyzed  pyocy- 
aneus culture,  there  is  added  from  three  to  five  grams  of  fresh,  finely 
divided  spleen  pulp.  This  mixture  is  treated  with  three  per  cent, 
of  potassium  carbonate  and  digested  at  37°.  During  this  proc- 
ess of  digestion  the  suspended  bits  of  spleen  substance  apparently 
undergo  a  process  of  agglutination,  resembling,  on  a  magnified  scale, 
the  phenomenon  which  may  be  seen  in  bacterial  cultures.  It  has 
been  observed  that  the  hay  bacillus  may  develop  in  this  mixture 
during  the  digestion  process  unless  trikresol  has  been  added.  Fi- 
nally the  spleen  pulp  is  apparently  digested,  while  only  small  bits  of 
capsule  and  fiber  remained  undissolved.  When  this  method  is  prop- 
erly carried  out  there  is  formed,  according  to  the  statements  of 
Emmerich  and  Low,  a  nuclease-immuneproteid  which  will  give  com- 
plete immunity  against  virulent  anthrax,  while  the  animal  thus 
treated  shows  no  elevation  of  temperature,  and  seems  to  remain  per- 
fectly well  in  every  respect. 

The  nuclease-immuneproteid  may  be  precipitated  by  the  addition 
of  ten  volumes  of  absolute  alcohol,  and  when  dried  in  vacuo  over 
sulphuric  acid  it  may  be  kept  for  an  indefinite  time  without  loss  of 
bacteriolytic  action.  Pyocyanase-immuneproteid  forms  a  homoge- 
neous greenish  or  yellowish  powder,  which  has  been  found  to  be  ser- 
viceable both  in  the  production  of  immunity  and  in  obtaining  cure, 
after  it  has  been  kept  for  two  years.  This  substance  is  said  to  keep 
better  if  about  5  per  cent,  of  dextrin  be  dissolved  in  the  bacteriolytic 
solution  before  precipitation  with  alcohol. 

The  importance  of  the  claims  of  Emmerich  and  Low  can  hardly 
be  overestimated,  provided  they  be  confirmed  by  other  careful  work- 
ers, and  be  found  to  be  generally  applicable  to  the  infectious  diseases. 
To  one  who  has  followed  the  above  given  account  of  their  investiga- 


IMMUNITY.  179 

tion,  it  must  be  evident,  provided  they  have  not  fallen  into  error 
somewhere,  that  they  have  succeeded  in  preparing  antitoxin  artifi- 
cially, and  if  their  statements  be  confirmed  it  must  be  admitted  that 
Ehrlich's  theory  concerning  the  nature  of  antitoxin  must  be  consider- 
ably modified.  Final  decision  on  this  matter  must  await  future  in- 
vestigations. 

When  animals  are  immunized  by  successive  treatments  with  a 
microorganism  or  its  products,  the  blood  serum  and  other  fluids  ob- 
tained from  the  body,  acquire  either  bactericidal  or  antitoxic  prop- 
erties. In  some  instances  the  immunity  secured  is  wholly  anti-bac- 
terial, while  in  others  it  is  antitoxic.  It  will  thus  be  seen  that  in  the 
production  of  artificial  immunity  we  may  expect  to  find  marked  dif- 
ferences depending  upon  the  microorganism  used,  and  the  kind  of 
animal  immunized.  We  will  first  discuss  bacterial  immunity.  The 
bactericidal  properties  possessed  by  the  fluids  of  the  body  of  the  im- 
munized animal  may  manifest  themselves  only  by  an  inhibitory  action 
on  the  growth  of  the  germ,  or  by  partially  depriving  it  of  its  capa- 
bility of  elaborating  toxins.  Early  in  his  investigations  of  this  sub- 
ject, Metschnikolf  found  that  anthrax  bacilli  grown  on  the  blood 
serum  of  sheep  immunized  to  this  disease  are  without  efi^ect  upon 
rabbits,  but  are  still  possessed  of  enough  vitality  to  induce  fatal 
anthrax  in  mice.  This  indicates  that  there  is  something  in  the  blood 
serum  of  the  immunized  sheep  which  reduces  the  virulence  of  the 
anthrax  bacillus.  In  some  instances  the  inhibitory  action  of  the 
immune  serum  manifests  itself  by  depriving  the  bacterium  of  its 
ability  to  produce  certain  of  its  characteristic  products.  Thus,  as 
has  already  been  stated,  Charrin  and  Roger  found  that  when  the 
bacillus  pyocyaneus  is  grown  in  the  blood  serum  of  animals  im- 
munized to  this  microorganism  it  no  longer  produces  its  character- 
istic coloring  matter.  The  inhibitory  action  of  the  body  fluids  may 
not  be  permanent,  and  when  the  bacillus  is  removed  from  their  direct 
influence  it  may  recover  all  of  its  ordinary  virulence.  Bordet  found 
that  when  streptococci  grown  in  the  serum  of  immunized  horses  were 
completely  freed  from  this  medium  they  are  as  virulent  as  those  de- 
veloped in  the  blood  serum  of  an  nnvaccinated  horse,  and  Roger  ob- 
served that  the  same  microorganisms  grow  well  in  the  serum  of  im- 
munized rabbits,  but  that  when  thus  grown  and  injected  into  other 
animals  along  with  some  of  the  immune  serum,  they  induce  only  a 
temporary  local  disturbance.  Next  he  inoculated  rabbits  with  normal 
streptococci  placed  in  the  blood  serum  of  immunized  animals  and 
found  that  the  rabbits  treated  with  this  mixture  remained  well,  while 
those  in  which  the  streptococci  and  the  serum  were  injected  into  dif- 
ferent parts  of  the  body  succumbed  to  the  infection. 

The  phenomenon  of  agglutination  is  a  manifestation  of  the  in- 
hibitory action  of  the  sera  of  immunized  animals  on  their  homologous 
bacteria.     The  fact  that  there  may  be  immunity  without  agglutina- 


180  IMMUNITY. 

tion,  and  vice  versa,  is  no  proof  that  this  phenomenon  is  not  a  result 
of  inhibitory  action  in  those  cases  in  which  it  does  occur.  It  should 
be  understood  that  agglutination  is  only  one  of  various  indications 
that  the  body  juices  of  immunized  animals  rob  their  homologous  bac- 
teria in  part  of  their  virulence.  Why  agglutination  does  not  take 
place  in  all  instances  we  are  not  as  yet  able  to  determine,  but  when 
it  does  occur  it  is  an  indication  that  the  blood  of  the  immunized  ani- 
mal has  some  detrimental  eflFect  upon  the  growth  and  virility  of  the 
microorganism.  It  is  true,  as  Metschnikoff  has  pointed  out,  that 
agglutination  is  not  uniformly  observed,  and  that  there  are  im- 
munized animals  which  furnish  sera  in  which  the  homologous  bac- 
teria grow  quite  normally,  but  this  observation  does  not  overthrow 
the  fact  that  agglutination  is  an  evidence  of  the  detrimental  effect  of 
the  sera  of  immunized  animals  on  the  bacteria  to  which  such  animals 
have  been  immunized. 

In  some  instances  the  body  juices  of  the  immunized  animal  mani- 
fest a  bactericidal  action  on  their  homologous  microorganisms,  but 
have  no  effect  on  their  toxins.  This  was  shown  to  be  the  case  by 
Metschnikoff  in  his  experiment  upon  the  coccobacillus  of  swine  plague. 
The  blood  sera  of  animals  immunized  to  this  bacterium  protect 
rabbits  against  infection,  but  furnish  no  protection  against  the  toxin. 
In  this  instance  we  have  to  do  with  purely  antiinfectious  phenomena. 
The  immunity  secured  is  antibacterial  and  not  antitoxic.  The  anti- 
infectious  properties  of  the  body  juices  are  not,  in  all  instances  at 
least,  strictly  specific.  The  sera  of  certain  immunized  animals  have 
a  bactericidal  action  not  only  on  their  homologous  bacteria,  but 
sometimes  on  closely  related  microorganisms.  Indeed  in  some  in- 
stances it  is  not  necessary  that  the  relationship  between  the  bacteria 
affected  in  like  manner  by  these  antiinfectious  sera  should  be  close. 
Thus,  as  has  already  been  stated  in  the  chapter  on  agglutinins,  the 
blood  serum  of  animals  immunized  to  the  cholera  vibrio  also  aggluti- 
nates several  other  vibrios,  and  it  has  been  shown  by  Issaeff  that  the 
blood  serum  of  animals  immunized  to  the  vibrio  of  Metschnikoff  has 
also  an  antiinfectious  action  on  the  pneumococcus.  It  has  also  been 
shown  that  normal  serum  may  in  some  instances  manifest  marked 
antiinfectious  properties.  For  instance,  one-tenth  cubic  centimeter 
of  the  blood  serum  of  a  healthy  man  suffices  to  protect  guinea-pigs 
against  cholera  peritonitis,  and  it  is  well  known  that  frequently  the 
blood  serum  of  men,  who  have  never  had  typhoid  fever,  agglutinates 
readily  the  bacillus  of  this  disease.  Not  only  is  this  the  case,  but 
the  blood  serum  of  such  men  may  protect  animals  against  the  perito- 
nitis normally  induced  in  them  by  the  typhoid  bacillus.  It  follows 
from  these  observations,  which  might  easily  be  multiplied,  that  the 
blood  of  normal  animals  frequently  contains  antiinfectious  substances. 

The  bacteriolytic  effect  is  the  most  marked  form  of  bactericidal 
action  that  has  been  observed  in  the  body  juices  of  artificially  im- 


IMMUNITY.  181 

munized  animals.  Of  course,  when  the  blood  serum  completely  dis- 
solves its  homologous  bacterium,  it  so  completely  destroys  it  that  it 
has  no  longer  either  infectious  or  toxic  properties.  It  has  been 
shown  quite  positively  that  agglutinins  and  lysins  are  not  identical, 
but  the  probabilities  are  that  they  represent  the  products  of  different 
stages  reached  in  the  process  of  immunization.  A  given  blood 
serum  may  have  an  agglutinating  action,  and  be  without  bacteriolytic 
effect,  or  vice  versa,  but  it  must  be  admitted  that  the  possession  of 
either  one  of  these  properties  by  the  body  juices  militates  against  the 
growth  of  the  infectious  agent,  and  consequently  each  must  be  con- 
sidered as  a  factor  in  the  production  of  artificial  immunity. 

The  question  concerning  the  origin  of  the  antiinfectious  substances 
in  the  production  of  artificial  immunity  is  one  concerning  which  at 
present  we  can  do  but  little  more  than  theorize.  The  weight  of  evi- 
dence seems  to  be  in  favor  of  the  view  that  by  successive  injections 
of  the  microorganism  or  its  products  the  leucocytes  are  stimulated 
to  increased  secretion  of  germicidal  substances.  Metschnikoff  claims 
that  it  is  a  general  rule  that  phagocytosis  is  more  pronounced  in  im- 
munized animals  than  in  those  not  immunized.  He  states  that 
when  a  microorganism  is  injected  into  an  animal  which  has  been 
immunized  to  this  germ,  the  phagocytes  of  the  animal  take  up  the 
invader  more  promptly  than  is  done  when  susceptible  animals  are 
inoculated  with  the  same  germ.  That  phagocytosis  is  more  marked 
in  immunized  animals  is  shown  by  introducing  the  microorganism  in 
localities  ordinarily  free  from  phagocytes,  such  as  the  subcutaneous 
tissue  and  the  anterior  chamber  of  the  eye.  When  this  experiment 
is  made,  the  phagocytes  collect  at  the  point  of  inoculation  very  much 
as  they  do  in  an  animal  possessed  of  natural  immunity,  and  there 
they  devour  the  invading  organism.  The  probabilities  certainly  are 
that  the  bactericidal  substances  found  in  the  serum  of  immunized 
animals  originate  in  the  phagocytes,  whose  capability  of  secreting 
this  substance  is  heightened  by  the  process  of  immunization.  Un- 
doubtedly it  is  true  that  phagolysis  also  plays  a  part  in  increasing 
the  antiinfectious  properties  of  the  body  juices  of  immunized  animals. 

We  now  turn  to  a  discussion  of  the  subject  of  immunity  to  bac- 
terial toxins.  Owing  to  the  fact  that  in  the  chapter  on  lysins  we 
have  developed  quite  fully  Ehrlich's  views  on  this  subject,  a  brief 
statement  will  be  all  that  will  be  necessary  at  this  place.  According 
to  Ehrlich,  we  are  to  consider  that  certain  cells  of  the  animal  body 
consist  of  a  nucleus,  which  he  designates  as  a  "  special  executive  cen- 
ter," from  which  there  radiate  many  side  chains.  It  must  be  under- 
stood that  this  is  a  chemical  and  not  a  morphological  conception  of 
the  cell.  These  side  chains  physiologically  are  concerned  in  processes 
of  nutrition.  Nutritive  substances  brought  to  the  cells  by  blood  or 
lymph  can  be  utilized  in  its  nutrition  only  after  they  have  entered 
into  combination  with  one  or  more  of  the  side  chains.     It  is  con- 


182  IMMUNITY. 

ceivable  that  some  of  these  side  chains  combine  with  carbohydrates, 
others  seize  upon  proteids,  and  others  still  may  fasten  themselves  to 
molecules  of  fat.  In  this  way  all  serve  in  securing  nutritive  material 
for  the  growth,  repair,  or  reproduction  of  the  cell.  It  is  also  possi- 
ble that  certain  pharmacological  agents  act  upon  the  diflPerent  tissues 
of  the  body  by  virtue  of  combinations  effected  with  these  cellular 
side  chains.  Now,  in  order  for  any  substance  to  be  poisonous  to  a 
given  animal,  there  must  be  in  the  body  of  that  animal  cells  with 
side  chains  capable  of  combining  with  the  substance.  A  body  which 
is  unable  to  effect  a  combination  with  any  animal  tissue  cannot 
be  poisonous.  It  is  not  supposed  that  cells  possess  side  chains 
especially  provided  for  the  purpose  of  combining  with  poisons,  but 
it  is  possible  that  certain  atomic  groups  which  ordinarily  are  con- 
cerned in  securing  nutritive  material  for  the  cell,  may  combine  with 
substances  which  are  capable  of  bringing  about  cellular  destruction. 
On  account  of  the  close  chemical  resemblance  between  the  bacterial 
toxins  and  certain  food  principles,  notably  those  proteid  in  character, 
this  supposition  seems  quite  plausible.  In  the  first  place,  then,  we 
may  say  that  the  body  cells  must  possess  toxophil  side  chains.  By 
this  we  mean  that  in  the  molecular  construction  of  certain  constit- 
uents of  the  cell  there  are  groups  of  atoms  which  may  combine  with 
bacterial  toxins.  In  the  second  place,  a  given  substance,  in  order  to 
be  toxic,  must  possess  certain  cytophil  side  chains.  Both  the  toxo- 
phil groups  of  the  cell,  and  the  cytophil  groups  of  the  toxin  may 
be  designated  as  haptophorous  bodies.  This  means  that  it  is  by  virtue 
of  these  groups  that  the  toxin  enters  into  combination  with  certain 
molecules  within  the  cell.  However,  the  toxin  is  possessed  not  only 
of  a  haptophorous,  but  also  of  a  toxophorous  group,  and  it  is  by  virtue 
of  the  latter  that  injury  to  the  cell  may  be  accomplished.  The  or- 
dinary nutritive  products  must,  according  to  this  theory,  possess 
cytophil  haptophorous  side  chains,  but  they  do  not  contain  the  tox- 
ophorous element.  Now  when  a  small  amount  of  a  bacterial  toxin  is 
introduced  into  the  body  of  a  susceptible  animal,  by  virtue  of  its 
haptophorous  group  it  combines  with  some  cell  in  the  body.  Whether 
or  not  it  causes  a  destruction  of  that  cell  depends  upon  the  immediate 
injury  done  by  this  combination.  If  the  toxin  be  introduced  in 
sufficiently  large  quantity,  it  may  completely  destroy  the  cell,  and  if 
a  sufficient  number  of  cells  be  destroyed,  death  will  result.  If,  on 
the  other  hand,  the  amount  of  toxin  introduced  be  relatively  small, 
and  the  number  of  toxophil  groups  in  the  cell  used  up  in  the  com- 
bination be  also  small,  the  cell  soon  recovers  from  the  slight  injury 
done  it,  but  recovers  deprived  of  the  side  chain  which  has  combined 
with  the  toxin.  Feeling  this  loss  and  needing  this  side  chain  in 
order  to  supply  itself  with  nutritive  material,  the  cell  throws  out  a 
new  side  chain,  and  when  stimulated  by  frequent  introduction  of 
small  quantities  of  toxin,  it  finally  not  only  makes  good  the  loss  with 


IMMUNITY.  183 

which  it  has  met,  but  throws  out  more  side  chains  than  it  can  possi- 
bly use.  In  other  words,  the  over-stimulated  cell  secretes,  as  it  were, 
toxophil  groups,  and,  as  frequently  happens  in  case  of  tissue  stim- 
ulation, the  process  continues  to  a  point  of  over-compensation. 
Finally  the  excess  of  toxophil  groups  thrown  out  by  the  cell  be- 
comes so  great  that  many  of  them  are  cast  oif  into  the  blood,  lymph, 
and  other  fluids  of  the  body.  These  cast-off  toxophil  groups  con- 
stitute the  antitoxin.  Now  if  a  toxin  be  introduced  into  the  body 
of  an  animal  whose  blood  and  lymph  are  filled  with  loose  toxophil 
groups,  the  toxin  is  neutralized  by  these  detached  side  chains,  and 
the  cell  escapes  injury  altogether.  In  this  way  Ehrlich  accounts  for 
the  formation  of  antitoxins  and  the  production  of  toxin  immunity. 
If  the  blood  serum  of  an  immunized  animal  be  injected  into  a  non- 
immune animal  and  the  latter  be  treated  with  the  homologous  toxin 
the  poison  thus  introduced  combines  with  the  antitoxin  dissolved  in 
the  body  juices,  and  the  cells  of  the  animal  thus  treated  wholly  escape 
any  injurious  effects.  It  may  be  well  at  this  point  to  make  the  fol- 
lowing quotation  from  Ehrlich's  own  statement  on  the  subject : 
"  The  theory  above  developed  allows  of  an  easy  and  natural  explana- 
tion of  the  origin  of  antitoxins.  In  keeping  with  what  has  already 
been  said,  the  first  stage  in  the  toxic  action  must  be  regarded  as  being 
the  union  of  the  toxin  by  means  of  its  haptophorous  group  to  certain 
side  chains  of  the  cell  protoplasm.  This  union  is,  as  animal  experi- 
ments with  a  great  number  of  toxins  show,  a  firm  and  enduring  one. 
The  side  chain  involved,  so  long  as  the  union  lasts,  cannot  exercise 
its  normal  nutritive  physiological  function — the  taking  up  of  definite 
food-stuffs.  It  is,  as  it  were,  shut  out  from  participating,  in  a 
physiological  sense,  in  the  life  of  the  cell.  We  are,  therefore,  now 
concerned  with  a  defect  which,  according  to  the  principles  so  ably 
worked  out  by  Professor  Carl  Weigert,  is  repaired  by  regeneration. 
These  principles,  in  fact,  constitute  the  leading  conception  in  my 
theory.  If,  after  union  has  taken  place,  new  quantities  of  toxin  are 
administered  at  suitable  intervals  and  in  suitable  quantities,  the  side 
chains  which  have  been  reproduced  by  the  regenerative  process  are 
taken  up  anew  into  union  with  the  toxin,  and  so  again  the  process 
of  regeneration  gives  rise  to  the  formation  of  fresh  side  chains.  In 
the  course  of  the  progress  of  typical  systemic  immunization,  as  this 
is  practised  in  the  case  of  diphtheria  and  tetanus  toxin  especially, 
the  cells  become,  so  to  say,  educated  or  trained  to  reproduce  the 
necessary  side  chains  in  ever-increasing  quantities.  As  Weigert  has 
confirmed  by  many  examples,  this,  however,  does  not  take  place  as  a 
simple  replacement  of  the  defect.  The  compensation  proceeds  far 
beyond  the  necessary  limit ;  indeed,  over-compensation  is  the  rule. 
Thus  the  lasting  and  ever-increasing  regeneration  must  finally  reach 
a  stage  at  which  such  an  excess  of  side  chains  is  produced  that,  to 
use  a  trivial  expression,  the  side  chains  are  present  in  too  great  a 


184  IMMUNITY. 

quantity  for  the  cell  to  carry,  and  are,  after  the  manner  of  a  secre- 
tion, handed  over  as  needless  ballast  to  the  blood.  Regarded  in  ac- 
cordance with  this  conception,  the  antitoxins  represent  nothing  more 
than  side  chains  reproduced  in  excess  during  regeneration,  and  there- 
fore pushed  off  from  the  protoplasm,  and  so  coming  to  exist  in  a  free 
state.  With  this  explanation,  the  phenomena  of  antitoxin  formation 
lose  all  their  strange,  one  might  say,  miraculous  characters." 

It  should  be  stated  that  according  to  Ehrlich's  theory  the  anti- 
infectious  substances  originate  in  the  body  of  the  inoculated  animal 
in  a  manner  similar  to  that  by  which  antitoxins  are  produced.  On 
this  point  he  makes  the  following  statement :  "  Much  more  complex 
than  in  the  cases  hitherto  discussed  are  the  conditions  when,  instead 
of  the  relatively  simple  metabolic  products  of  microbes,  the  living 
microorganisms  themselves  come  to  be  considered ;  as  in  immunization 
against  cholera,  typhoid,  anthrax,  swine  fever,  and  many  other  infec- 
tious diseases.  There  then  come  into  existence  alongside  of  the  anti- 
toxins produced  as  a  result  of  the  action  of  the  toxins,  manifold  other 
reaction  products.  This  is  because  the  bacterium  is  a  highly  com- 
plicated living  cell,  of  which  the  solution  in  the  organism  yields  a 
great  number  of  bodies  of  different  nature,  in  consequence  of  which 
a  multitude  of  antibodies  are  called  into  existence.  Thus  we  see  as 
a  result  of  the  injection  of  bacterial  cultures,  that  there  arise  along- 
side of  the  specific  bacteriolysins,  which  dissolve  the  bacteria,  other 
products,  as,  for  example,  '  coagulins,'  i.  e.,  substances  which  are  able 
to  cause  the  precipitation  of  certain  albuminous  bodies  contained  in 
the  culture  fluid  injected ;  also  the  so  much  discussed  '  agglutinins,' 
the  antiferments,  and  no  doubt  many  other  bodies  which  we  have 
not  yet  recognized.  It  is  by  no  means  unlikely  that  each  of  these 
reaction  products  finds  its  origin  in  special  cells  of  the  body ;  on  the 
other  hand,  it  is  quite  likely  that  the  formation  of  any  single  one  of 
these  bodies  is  not  of  itself  sufficient  to  confer  immunity.  Thus  in 
case  of  the  introduction  of  bacteria  into  the  body,  we  have  to  do  with 
a  many-sided  production  of  different  forms  of  '  antibodies,'  each  of 
which  is  directed  only  against  one  definite  quality  or  metabolic  product 
of  the  bacterial  cell.  Accordingly,  in  recent  times,  the  practice  of 
using  for  the  production  of  immunization  definite  toxic  bodies  isolated 
from  the  bacterial  cell  has  been  more  and  more  given  up,  and  for  this 
purpose  it  is  now  regarded  as  important  to  employ  the  bacterial  cells 
as  intact  as  possible." 

According  to  Ehrlich's  theory,  antitoxins  are  not  modified  toxins, 
but  are  the  products  of  certain  cells  of  the  animal  body,  and  are  pro- 
duced by  the  stimulation  of  these  cells  by  the  toxin.  If  it  can 
be  shown  that  antitoxins  have  their  origin  in  the  toxin  molecule, 
Ehrlich's  theory  will  be  overthrown.  The  evidence  against  the 
theory  that  antitoxins  are  modified  toxins  is  well-nigh  conclusive,  and 
may  be  stated  as  follows  :  (1)  The  taking  of  large  quantities  of  blood. 


IMMUNITY.  185 

one  third  or  more  of  the  contents  of  the  body,  from  immunized  ani- 
mals, at  intervals  of  time  sufficiently  long  to  allow  the  animal  to  re- 
cover to  such  an  extent  that  its  health  is  not  seriously  impaired,  does 
not  diminish  the  immunity  of  this  animal  as  markedly  as  would  be 
expected  if  the  antitoxin  was  a  modified  toxin,  and  the  quantity  of 
the  former  could  not  be  greater  than  the  quantity  of  the  latter  em- 
ployed in  immunizing  the  animal.  (2)  If  an  immunized  animal  be 
bled  to  death,  and  its  vascular  system  be  washed  with  physiological 
salt  solution  until  all  the  blood  that  can  possibly  be  washed  out  is 
removed,  yet  infusions  of  certain  tissues  of  this  animal  contain  anti- 
toxin. It  must,  however,  be  granted  that  the  tissues  may  retain 
some  of  the  modified  toxin  in  the  form  of  antitoxin  after  the  most 
thorough  washing  out  of  the  vascular  system.  (3)  The  quantity  of 
antitoxin  obtained  is  not  always  at  least  in  direct  proportion  to  the 
amount  of  toxin  used  in  the  production  of  the  immunity,  as  would 
necessarily  be  the  case  if  the  antitoxin  originated  in  the  toxin.  Cer- 
tainly it  must  be  admitted  that  if  the  antitoxin  comes  from  the  toxin, 
the  amount  of  the  former  produced  can  never  be  greater  than  the 
quantity  of  the  latter  used  in  its  production.  In  other  words,  a  part 
can  never  be  greater  than  the  whole. 

It  has  been  claimed  by  some  that  toxins  can  be  converted  into 
antitoxins  by  the  action  of  electricity.  In  our  last  edition  we  made 
the  following  statement  concerning  this  claim  :  "  Smirnow  has  writ- 
ten quite  at  length  to  show  that  toxins  may  be  converted  into  anti- 
toxins by  the  long-continued  action  of  an  electric  current.  We  fail 
to  find  in  his  recorded  experiments  any  justification  of  his  claim. 
He  makes  quite  a  point  of  the  fact  that  during  the  continuance  of 
the  electrolysis  the  bouillon  becomes  more  deeply  colored  at  one  pole 
and  nearly  decolored  at  the  other.  Now,  the  merest  tyro  in  physio- 
logical chemistry  knows  that  acids  deepen  the  color  of  beef  tea,  urine, 
or  any  other  fluid,  the  coloring  matter  of  which  is  derived  from 
hemoglobin.  So  much  for  his  chemistry.  His  physiology  is  worse. 
When  he  administers  this  artificial  antitoxin  in  too  large  doses  he 
kills  his  animals  ;  and  of  what  do  they  die  ? — of  diphtheria.  He 
has  only  modified  and  reduced  the  virulence  of  his  diphtheria  toxin 
by  the  acid  generated  by  the  electrolysis  of  the  inorganic  salts  in 
the  bouillon.  Toxins  may  be  convertible  into  antitoxins,  and  elec- 
tricity may  be  the  agent  capable  of  inducing  this,  but  Smirnow  has 
proved  neither  one  nor  the  other." 

Since  the  above  was  written  other  investigators  have  reported  the 
confirmation  of  Smirnow's  results,  but  after  a  careful  study  of  these 
reports  we  see  no  reason  for  changing  the  opinion  expressed  above. 
We  will  investigate  only  one  of  these  reports.  Bolton  and  Pease  ^ 
passed  electric  currents  through  bouillon  solutions  of  diphtheria 
toxin  and  found  that  5  c.c.  of  the  product  from  the  positive  pole 
^  Transactions  of  the  Association  of  American  Physicians,  11,  1896. 


186  IMMUNITY. 

mixed  with  1  c.c.  of  diphtheria  toxin,  containing  ten  fatal  doses,  in- 
jected into  a  guinea-pig  did  not  cause  death,  and  from  this  they  con- 
clude that  they  have  converted  the  toxin  into  antitoxin.  They  state 
that  during  the  electrolytic  progress  a  peculiar  odor  resembling  that 
of  chlorid  of  lime  is  given  off,  "  but  it  was  found  that  the  fluid  con- 
tained only  one-tenth  of  one  per  cent,  of  chlorin."  They  made  no 
control  experiment  in  order  to  determine  whether  or  not  this  amount 
of  chlorin  will  destroy  the  quantity  of  diphtheria  toxin  employed  by 
them,  and  until  such  an  experiment  has  been  made  we  will  be  in- 
clined to  the  opinion  that  the  5  c.c.  of  electrolyzed  solution  contained 
enough  chlorin  not  only  to  destroy  its  own  toxin,  but  also  that  in  the 
1  c.c.  of  non-electrolyzed  toxin  with  which  it  was  mixed. 

It  must  be  admitted  that  it  is  within  the  range  of  possibility  that 
antitoxins  may  contain  a  ferment  derived  from  the  toxin,  and  which 
in  the  animal  body  during  the  process  of  immunization  has  com- 
bined with  the  constituents  of  certain  tissues.  This  view  is  in  ac- 
cord with  the  experimental  observations  of  Emmerich  and  Low, 
which  we  have  already  detailed. 

Centanni  endeavored  to  determine  experimentally  what  organ  or 
tissue  of  the  body  is  affected  in  the  production  of  immunity  against 
rabies.  His  work  consisted  in  endeavoring  to  induce  immunity  in 
a  second  series  of  rabbits  by  treating  them  subcutaneously  with  the 
blood  serum  and  with  emulsions  made  from  the  various  organs  of  the 
members  of  a  primary  series,  already  rendered  partially  or  wholly 
immune  by  the  ordinary  method.  In  doing  this  he  used  amounts  of 
each  tissue  proportional  to  the  weight  of  the  animal ;  thus  the  central 
nervous  system  of  an  average  rabbit  is  equal  in  weight  to  one  three 
hundredth  part  of  the  total  body  weight  of  the  animal,  and  the  total 
blood  of  the  same  animal  furnished  an  amount  of  serum  equal  in 
weight  to  one  one  hundred  and  fiftieth  part  of  its  body  weight. 
Therefore  he  injected  into  a  rabbit  weighing  1,800  grams,  six  grams 
of  nervous  tissue  made  into  an  emulsion,  and  into  another  of  the  same 
weight,  12  grams  of  blood  serum  taken  from  rabbits  already  made 
immune,  and  then  tested  the  immunity  of  the  members  of  this  second 
series.  Emulsions  of  other  tissues  were  employed  in  proportionately 
the  same  doses.  By  this  procedure  he  found,  as  he  thinks,  that  in 
the  production  of  immunity  against  rabies,  the  immunizing  substance 
is  stored  up  in  the  central  nervous  system.  Moreover  he  found  that 
the  immunizing  substance  remained  in  the  nervous  system  long 
after  it  had  disappeared  from  the  blood  and  other  organs.  Thus  it 
would  seem  that  Pasteur  hit  upon  the  right  thing  exactly  when  he 
selected  emulsions  of  the  spinal  cord  as  the  proper  material  with 
which  he  could  best  induce  immunity  against  hydrophobia.  From 
these  experiments,  Centanni  drew  the  general  conclusion  that  in  the 
production  of  immunity  against  any  diseases,  the  immunizing  sub- 
stance is  stored  up  in  greatest  quantity  and  most  permanently  in 


IMMUNITY.  187 

that  organ  or  tissue  most  seriously  affected  by  the  disease.  The  ex- 
periments made  by  Roux  show  quite  conclusively  that  the  tetanus 
toxin  combines  with  brain  cells  and  it  may  be  said  that  this  is  a  par- 
tial confirmation  of  the  general  law  which  Centanni  attempted  to 
formulate. 


CHAPTER  X. 

FOOD  POISONING  (Bromatotoxismus  i). 

Untoward  results  frequently  follow  the  eating  of  food  which  is 
ordinarily  harmless.  These  ill  effects  may  be  due  to  the  following 
causes  :  (1)  Grains  may  become  infected  with  the  poisonous  parasitic 
fungi,  such  as  ergot.  (2)  Both  plants  and  animals  may  feed  upon 
substances  which  are  not  harmful  to  them  but  which  may  seriously 
affect  man  on  account  of  his  greater  susceptibility ;  it  is  said  that 
birds  which  have  fed  upon  mountain  laurel  furnish  food  poisonous  to 
man.  (3)  The  flesh  of  some  animals  is  poisonous  during  the  period 
of  physiological  activity  of  certain  glands  ;  this  is  true  of  certain  fish 
during  the  spawning  season.  (4)  Any  food  may  be  infected  with 
specific  germs  and  serve  as  the  carrier  of  infection ;  typhoid  fever  is 
frequently  disseminated  in  this  manner.  (5)  The  animal  may  be 
afflicted  with  a  specific  disease,  and  this  may  be  transmitted  to  man 
in  the  meat  or  milk ;  tuberculosis  may  be  spread  in  this  way.  (6) 
Foods  of  various  kinds  may  become  contaminated  with  saprophytic 
bacteria,  which  by  their  growth  elaborate  chemical  poisons  either 

^  The  following  is  a  glossary  of  the  new  words  employed  in  this  article  : 

Bromatotoxismus,  fipuua  {ppu/iarog),  food,  and  to^ck6v,  poison.  Food  poisoning 
or  poisoning  with  food. 

Bromatotoxicon.     A  general  term  for  the  active  agent  in  a  poisonous  food. 

Bromatotoxin.  A  basic  poison  generated  in  food  by  the  growth  of  bacteria  or 
fungi. 

Galactotoxismus,  yaAa  (yaAaKTog),  milk.     Milk  poisoning. 

Galactotoxicon. 

Galactotoxin. 

Ichthyotoxismus,  ix^'i'?,  fish.     Fish  poisoning. 

Ichthyotoxicon. 

Ichthyotoxin. 

Kreotoxismus,  Kpkig  or  Kpeug,  meat.     Meat  poisoning. 

Kreotoxicon. 

Kreotoxin. 

Mytilotoxismus,  fivTiXog,  a  sea-mussel.  Mussel  poisoning.  Used  already  by 
Husemann. 

Mytilotoxicon. 

Mytilotoxin.  The  name  given  by  Brieger  to  the  ptomain  discovered  by  him  in 
poisonous  mussel. 

Sitotoxismus,  airoc,  cereal  food.     Poisoning  with  vegetable  food. 

Sitotoxicon. 

Sitotoxin. 

Tyrotoxismus,  rvpog^  cheese.     Cheese  poisoning.     Used  already  by  Husemann. 

Tyrotoxicon. 

Tyrotoxin. 

Husemann  uses  the  word  zootrophotoxismus  to  indicate  poisoning  with  animal 
food.  The  same  author  has  employed  the  word  halichthyotoxismus  to  designate 
poisoning  with  fisli. 

188 


MYTILOTOXISMUS.  189 

before  or  after  the  food  has  been  eaten ;  this  is  the  most  common 
form  of  food  poisoning. 

Mytilotoxismus.— Judging  from  the  symptoms  induced,  there 
seem  to  be  three  kinds  of  poisonous  mussel.  In  some  cases  the  symp- 
toms resemble  those  induced  by  a  gastro-intestinal  irritant.  For- 
dere  reports  the  case  of  a  soldier,  who,  after  eating  a  large  dish  of 
mussels,  suffered  from  nausea,  vomiting,  pain  in  the  stomach,  tenes- 
mus, and  rapid  pulse ;  after  death,  which  occurred  within  two  days, 
the  stomach  and  intestines  were  found  inflamed  and  filled  with  tena- 
cious mucus.  Combe  and  others  also  reported  cases  of  the  choleraic 
form  of  mussel  poisoning. 

However,  the  symptoms  most  frequent  in  man  after  the  eating  of 
poisonous  mussels  are  more  purely  nervous.  A  sensation  of  heat 
and  itching  appears,  usually  in  the  eyelids,  and  soon  involves  the 
whole  face,  and  perhaps  a  large  portion  of  the  body.  An  eruption 
usually  called  nettle  rash,  though  it  may  be  papular  or  vesicular, 
covers  the  parts.  The  itching  is  most  annoying,  and  may  be  ac- 
companied by  marked  swelling.  Often  there  is  asthmatic  breathing, 
which  is  relieved  only  by  ether.  In  some  cases  reported  by  Mohr- 
ing  dyspnoea  preceded  the  eruption,  the  patients  became  insensible, 
the  face  livid,  and  convulsive  movements  of  the  extremities  were 
noticed.  Burrows  reports  similar  cases  with  convulsive  tremors, 
coma,  and  death  within  three  days. 

In  a  third  class  of  cases  there  may  be  observed  intoxication  re- 
sembling that  of  alcohol,  followed  by  paralysis,  coma  and  death. 

In  1827,  Combe  observed  thirty  persons  poisoned,  two  of  them 
fatally,  with  mussels.  He  described  the  symptoms  as  follows : 
"  None,  so  far  as  I  know,  complained  of  anything  peculiar  in  the 
smell  or  taste  of  the  animals  and  none  suffered  immediately  after 
taking  them.  In  general,  an  hour  or  two  elapsed,  sometimes  more ; 
and  the  bad  effects  consisted  rather  in  uneasy  feelings  and  in  de- 
bility than  in  any  distress  referable  to  the  stomach.  Some  children 
suffered  from  eating  only  two  or  three  ;  and  it  will  be  remembered 
that  Robertson,  a  young  and  healthy  man,  only  took  five  or  six.  In 
two  or  three  hours  they  complained  of  a  slight  tension  of  the  stom- 
ach. One  or  two  had  cardialgia,  nausea  and  vomiting  ;  but  these 
were  not  general  or  lasting  symptoms.  They  then  complained  of  a 
prickly  feeling  in  their  hands,  feet,  and  constriction  of  the  mouth 
and  throat,  difficulty  of  swallowing,  and  speaking  freely,  numbness 
about  the  mouth,  gradually  extending  to  the  arms,  with  great  de- 
bility of  the  limbs.  The  degree  of  muscular  debility  varied  a  good 
deal,  but  was  an  invariable  symptom.  In  some  it  merely  prevented 
them  from  walking  firmly,  but  in  the  most  of  them  it  amounted  to 
perfect  inability  to  stand.  While  in  bed  they  could  move  their 
limbs  with  tolerable  freedom,  but  on  being  raised  to  the  perpendicu- 
lar posture  they  felt  their  limbs  sink  under  them.     Some  complained 


190  FOOD  POISONING. 

of  a  bad,  coppery  taste  in  their  mouths,  but  in  general  this  was  in 
answer  to  what  lawyers  call  a  '  leading  question.'  There  was  slight 
pain  in  the  abdomen,  which  increased  on  pressure,  particularly  in 
the  region  of  the  bladder,  which  organ  suffered  variously  in  its 
functions.  In  some  the  secretion  of  urine  was  suspended,  in  others 
it  was  free,  but  passed  with  pain  and  great  effort.  The  action  of 
the  heart  was  feeble ;  the  breathing,  unaffected ;  the  face  pale,  ex- 
pressive of  much  anxiety  ;  the  surface,  rather  cold  ;  the  mental  fac- 
ulties, unimpaired.  Unluckily  the  two  fatal  cases  were  not  seen  by 
any  medical  person,  and  we  are,  therefore,  unable  to  state  minutely 
the  train  of  symptoms.  We  ascertained  that  the  woman,  in  whose 
house  were  five  sufferers,  went  away  as  in  a  gentle  sleep,  and  that  a 
few  minutes  before  death  she  had  spoken  and  swallowed." 

The  woman  mentioned  by  Combe  died  within  three  hours,  and  the 
other  death  was  that  of  a  watchman,  who  was  found  dead  in  his  box 
six  or  seven  hours  after  he  had  eaten  of  the  mussels.  Post-mortem 
examination  of  these  showed  no  abnormality  ;  the  stomach  contained 
some  of  the  food  partially  digested.  The  explorer  Vancouver  re- 
ports four  cases  similar  to  those  observed  by  Combe ;  one  of  the 
sailors  died  in  five  and  one-half  hours  after  eating  the  mussels. 

Schmidtmann  has  reported  cases  observed  by  himself  in  some 
workmen  and  members  of  their  families,  who  had  partaken  of  mus- 
sels taken  near  a  newly  constructed  dock.  The  symptoms  appeared, 
according  to  the  amount  eaten,  from  soon  after  eating  to  several 
hours  later.  There  was  a  sensation  of  constriction  in  the  mouth, 
throat  and  lips.  The  teeth  were  set  on  edge  as  though  sour  apples 
had  been  eaten.  There  was  no  headache,  a  sensation  of  flying  and 
an  intoxication  similar  to  that  produced  by  alcohol.  The  pulse  was 
hard  and  rapid  ;  no  elevation  of  temperature  ;  the  pupils  were  dilated 
and  reactionless.  Speech  was  difficult,  broken  and  jerky.  The 
limbs  felt  heavy ;  the  patients  grasped  spasmodically  at  objects  and 
missed  their  aim.  The  legs  were  no  longer  able  to  support  the  body. 
The  knees  knocked  together.  There  was  nausea,  vomiting,  no  ab- 
dominal pain,  no  diarrhoea.  The  hands  began  to  feel  cold.  The 
sensation  of  cold  soon  extended  over  the  entire  body,  and  in  some 
the  perspiration  flowed  freely.  There  was  a  feeling  of  suffocation, 
then  a  restful  and  dreamless  sleep.  One  person  died  in  one  and 
three-quarter  hours,  another  in  three  and  one-half  hours,  and  a  third 
in  five  hours,  after  eating  of  the  mussels.  In  one  of  these  fatal 
cases  rigor  mortis  was  marked  and  remained  for  twenty-four  hours. 
The  vessels  of  all  the  organs  were  distended,  only  the  heart  was 
empty.  There  was  marked  hyperemia  and  swelling  of  the  mucous 
membrane  of  the  stomach  and  intestines,  and  the  spleen  was  enor- 
mously enlarged,  and  the  liver  showed  numerous  hemorrhagic  infarc- 
tions. 

Many  theories  have  been  advanced  to  account  for  poisonous  mus- 


MYTILOTOXISMUS.  191 

sels.  It  was  formerly  believed  that  the  efiPects  were  due  to  the  cop- 
per which  the  animals  obtained  from  the  bottoms  of  vessels,  but  as 
Christison  remarks,  copper  does  not  produce  these  symptoms.  More- 
over, Christison  made  analysis  of  the  mussels  which  produced  the 
symptoms  observed  by  Combe,  and  was  unable  to  detect  any  copper. 
Bouchardat  found  copper  in  some  poisonous  mussels,  but  he  does  not 
state  the  amount  of  the  metal  nor  the  source  of  the  animals.  Edwards 
advanced  the  theory  that  the  symptoms  were  wholly  due  to  idiosyn- 
crasy of  the  consumer.  This  certainly  is  not  a  tenable  hypothesis 
in  such  instances  as  those  reported  by  Combe  and  Schmidtmann, 
where  a  large  number  or  all  those  who  partook  of  the  food  were 
affected.  Coldstream  stated  that  the  livers  of  poisonous  mussels  are 
larger,  darker,  and  more  brittle  than  normal,  and  these  changes  he 
believes  are  due  to  a  diseased  condition  of  the  animals.  Many  have 
supposed  that  the  poisonous  effects  were  due  to  a  peculiar  species  of 
medusa  upon  which  the  mussels  feed,  and  De  Beume  found  in  the 
vomited  matter  of  one  person  some  medusse  and  he  states  that  these 
are  most  abundant  during  the  summer,  when  mussels  are  most  fre- 
quently found  to  be  poisonous.  The  theory  of  Burrows  that  mussels 
are  always  poisonous  during  the  period  of  reproduction  at  one  time 
received  considerable  credit ;  however,  cases  of  poisoning  have  oc- 
curred at  different  seasons  of  the  year.  In  1872,  Crumpe  suggested 
that  there  is  a  species  of  mussel  which  is  in  and  of  itself  poisonous, 
and  this  species  is  often  mixed  with  the  edible  variety.  It  has  been 
stated  that  the  poisonous  species  has  a  brighter  shell,  a  sweet,  more 
penetrating,  bouillon-like  odor  than  the  non-poisonous  ;  also  that  the 
flesh  of  the  former  is  yellow  and  that  the  water  in  which  they  are 
cooked  is  bluish.  This  theory,  however,  is  opposed  by  the  majority 
of  zoologists.  M5bius  states  that  the  peculiarities  of  the  supposed 
poisonous  variety  pointed  out  by  Virchow  and  Schmidtmann  are 
really  due  to  the  conditions  under  which  the  animals  live,  the  amount 
of  salt  in  the  water,  the  temperature  of  the  water,  whether  it  is 
moving  or  still  water,  the  nature  of  the  bottom,  etc.  He  also  states 
that  the  sexual  glands,  which  form  the  greater  part  of  the  mantle,  are 
white  in  the  male  and  yellow  in  the  female.  The  theory  of  a  poison- 
ous species  has  been  abandoned  since  it  has  been  shown  that  edible 
mussels  may  become  poisonous  if  left  in  filthy  water  fourteen  days  or 
longer,  and,  on  the  other  hand,  poisonous  ones  may  become  fit  for 
food  if  kept  for  four  weeks  in  good  water. 

Cats  and  dogs  which  have  eaten  voluntarily  of  poisonous  mussels 
have  suffered  from  symptoms  similar  to  those  observed  in  man ;  and 
rabbits  have  been  poisoned  by  the  administration  of  the  water  in 
which  the  food  has  been  cooked.  A  rabbit  treated  in  this  manner 
by  Schmidtmann  died  within  one  minute.  From  these  mussels 
Brieger  extracted  the  ptomain  mytilotoxin,  which  will  be  discussed 
in  a  subsequent  chapter.     Whether  or  not  those  mussels  which  pro- 


192  FOOD  POISONING. 

duce  other  symptoms  also  contain  ptomains  remains  for  future  in- 
vestigations to  determine. 

In  1887,  three  cases  of  mussel  poisoning,  one  fatal  case,  occurred 
at  Wilhelmshaven,  the  place  which  supplied  Brieger  with  the  mus- 
sels from  which  he  obtained  mytilotoxin.  Schmidtmann  found  that 
non-poisonous  mussels  placed  in  the  waters  of  this  bay  soon  became 
poisonous,  and  that  the  poisonous  mussels  from  the  bay  placed  in 
the  open  sea  soon  lose  their  toxic  properties.  Linder  has  found  in 
the  water  of  the  bay  and  in  the  mussels  living  in  it  a  great  variety 
of  protozoa,  amoebae,  bacteria  and  other  organisms,  which  are  not  found 
in  the  water  of  the  open  sea  nor  in  the  non-poisonous  mussels.  He 
also  ascertained  that  if  the  water  of  the  bay  be  filtered,  non-poisonous 
mussels  placed  in  it  do  not  become  poisonous,  and  he  concludes  that 
poisonous  mussels  are  those  which  are  sufiering  from  disease  due 
to  residence  in  filthy  water.  Cameron  makes  a  somewhat  similar 
statement  about  the  poisonous  mussels  near  Dublin,  taken  from 
water  contaminated  with  sewage.  He  found  that  the  livers  of  these 
animals  were  much  enlarged,  and  from  them  he  obtained  a  base  that 
is  probably  identical  with  mytilotoxin.  That  oysters  taken  from 
beds  near  the  outlet  of  sewers  may  be  contaminated  with  the  speci- 
fic germ  of  typhoid  fever  has  been  well  demonstrated,  within  the 
past  few  years,  and  that  they  may  become  poisonous  in  the  same 
way  that  mussels  acquire  harmful  properties  is  also  well  known. 
Pasquier  reported  cases  of  poisoning  at  Havre  from  the  eating  of 
oysters  taken  from  an  artificial  bed  near  the  outlet  of  a  drain  from  a 
public  water  closet.  Christison  says  that  an  unusual  prevalence  of 
colic,  diarrhoea  and  cholera  at  Dunkirk  was  believed  to  have  been 
traced  to  an  importation  of  oysters  from  the  Normandy  coast.  There 
should  be  police  regulations  against  the  sale  of  all  kinds  of  mollusks, 
and  all  kinds  of  fish  as  well,  taken  from  filthy  water.  Special  at- 
tention should  be  given  to  localities  that  have  once  supplied  poison- 
ous food  of  this  kind.  Many  popular  rules  have  been  formulated 
for  the  easy  recognition  of  poisonous  mussels,  and  to  some  of  these 
credence  has  been  given  by  medical  authors.  An  unusually  large 
mussel  is  regarded  with  suspicion,  and  Lohmeyer  gives  measure- 
ments that  may  guide  the  person  in  search  of  this  article  of  food. 
Stress  is  placed  on  color  by  some,  and  one  is  advised  to  avoid  the 
dark  brown-blue,  and  purchase  the  dark-blue  or  dark  green-blue. 
We  may  expect  to  see  the  prudent  hungry  man  draw  from  his 
pocket  a  scale  of  colors  and  carefully  compare  it  with  the  shell  of 
the  juicy  bivalve  before  he  consigns  it  to  his  digestive  organs,  if  he 
is  to  observe  the  rules  laid  down  in  some  recent  medical  works. 
Then  he  will  take  the  dimensions  of  the  whole,  measure  the  thick- 
ness of  its  shell,  and  then  its  strength,  for  we  are  informed  that  the 
poisonous  clam  has  a  thin,  brittle  shell.  Seriously,  one  is  to  avoid 
shellfish  from  impure  water,  and  he  may  properly  insist  that  they 


ICHTHYOTOXISMUS.  193 

be  washed  in  clean  water,  and  certainly  one  should  avoid  eating  this 
kind  of  food  when  it  has  stood  for  a  few  hours  at  summer  heat  in 
the  form  of  broth. 

Ichthyotoxismus. — Some  fish  are  always  poisonous  ;  others  are 
poisonous  only  during  the  spawning  season,  and  still  others  are  sub- 
ject to  epidemic  bacterial  diseases,  and  those  affected  with  certain  of 
these  diseases  furnish  flesh  that  is  toxic  to  man,  or,  in  other  words, 
the  bacterial  disease  is  transmitted  to  man  with  his  food.  Lastly, 
fish,  like  other  kinds  of  meat,  may  become  infected  with  saprophytic 
germs  which  produce  toxins  poisonous  to  man.  The  Spaniards  use 
the  word  siguatera  ^  to  designate  the  complex  of  symptoms  induced 
in  man  by  the  eating  of  fish  that  are  physiologically  poisonous  ;  and 
Blanchard  proposes  the  general  adoption  of  this  term,  while  he 
suggests  that  the  word  botulismus  or  botulism  be  used  to  designate 
diseased  conditions  which  result  from  the  eating  of  any  kind  of  meat 
that  is  harmful  on  account  of  bacterial  infection.  His  statement  is 
substantially  as  follows  :  "  There  are  two  distinct  categories  of  in- 
toxication with  the  flesh  of  vertebrates  : 

"  Botulismus  is  an  intoxication  induced  by  meat  invaded  by  mi- 
crobes and  containing  the  ptomains  elaborated  by  them  ;  this  term 
is  applicable  not  only  to  disease  caused  by  market  meat,  but  also  to 
that  induced  by  preserved  foods, 

''Siguatera  is  an  intoxication  caused  by  fresh  food,  not  infected 
by  bacteria,  and  in  which  the  poisonous  principles  are  leucomains 
formed  by  the  physiological  activity  of  the  tissues.  I  propose  to 
designate  this  category  of  intoxication  by  the  word  siguatera,  a  name 
employed  by  the  Spanish  physicians  of  the  Antilles  to  indicate 
poisoning  by  eating  fish." 

While  we  have  not  accepted  Blanchard's  nomenclature  as  appli- 
cable to  all  kinds  of  poisonous  meats,  the  distinction  made  by  him 
admirably  states  the  differences  in  the  two  kinds  of  fish  poisoning. 
It  is  a  question  whether  or  not  we  shall  discuss  in  this  connection 
those  fish  whose  flesh  is  not  harmful,  but  which  are  supplied  with 
poisonous  glands.  However,  as  the  secretions  of  the  special  glands 
owe  their  toxic  properties  to  physiological  poisons,  we  will  include 
them  in  this  category  and  will  make  brief  mention  of  them. 

Kobert  makes  the  following  classification  of  poisonous  fish  : 

1.  Many  fish  possess  poisonous  glands  that  are  connected  with 
their  barbed  fins  with  which  they  wound  their  enemies.  The 
structure  of  these  glands  is  similar  to  those  of  poisonous  snakes. 
After  the  removal  of  the  skin  containing  these  glands  the  flesh  is  not 
poisonous.  Such  are  Trachinus  draco  of  the  German  lakes,  and 
Serranus  scriba  of  the  Mediterranean  sea.  Stomias  boa  is  feared  on 
account  of  its  bite ;  many  roaches  have  a  poisonous  barb  in  the  tail. 
Bottard  describes  five  classes  of  fish  supplied  with  poisonous  glands. 
^  Pronounced  sig-wah-te'ra. 
13 


194  FOOD  POISONING. 

(1)  Of  this  class  Synanceia  brachio  is  a  type  and  has  its  poison  ap- 
paratus in  the  dorsal  fin,  consisting  of  thirteen  barbs,  each  of  which 
has  two  poison  reservoirs.  Each  of  the  twenty-six  reservoirs  is 
supplied  with  ten  or  twelve  tubular  glands,  the  secretion  of  which 
is  a  clear,  bluish,  feebly  acid  fluid.  Undiluted,  this  solution  causes 
local  gangrene ;  diluted,  it  induces  paralysis.  In  Plotosus  lanceatus 
in  front  of  the  ventral  fin  there  is  a  hollow  barb  with  closed  end, 
connected  with  a  poison  reservoir,  and  the  fluid  flows  only  when  the 
barb  is  broken.  (2)  Trachinus  draco  is  a  typical  example  of  this 
class.  The  apparatus  of  Cottus  scorpio  and  C  bubalis  also  is  of 
this  kind.  There  are  three  hollow  barbs  on  the  gill  cover  and  the 
reservoirs  connected  with  this  secrete  a  poison  only  during  the 
spawning  season.  (3)  Thalassophryne  reticulata  has  two  hollow 
barbs,  one  on  the  gill  cover  and  the  other  on  the  back.  (4)  Mureua 
helena  has  on  the  gills  an  open  pocket,  the  walls  of  which  are  lined 
with  cells  secreting  a  poison  that  moistens  the  teeth.  (5)  Scorpena 
scropha  and  S.  porcus  have  open  poison  glands  connected  with  hol- 
low barbs  set  in  the  dorsal  and  ventral  fins.  Chemically  nothing  is 
known  of  the  nature  of  these  poisons  ;  pharmacologically  it  has  been 
demonstrated  that  they  cause  severe  inflammation  of  the  subcuta- 
neous tissue. 

Trachinus  draco,  ordinarily  known  as  the  "dragon-weaver"  or 
"  sea-weaver,"  is  one  of  the  best  known  of  the  fish  possessed  of 
poisonous  barbs.  The  varieties  of  this  species  are  widely  distributed 
in  salt  waters.  It  is  a  handsome  fish,  somewhat  resembling  the 
trout,  and  marked  with  blue  and  brown  stripes.  While  bathing, 
men  sometimes  wound  their  feet  with  the  barbs  of  this  fish  which 
lies  half  buried  in  the  sand.  It  also  happens  that  fishermen  some- 
times incautiously  prick  their  fingers  with  these  barbs.  Almost  im- 
mediately there  are  knife-like  pains  felt  about  the  wound  and  these 
quickly  extend  over  the  body.  Cardialgia  may  be  most  excruciat- 
ing and  there  is  a  sensation  of  suffocation.  The  forehead  is  covered 
with  profuse  cold  perspiration,  and  the  heart  becomes  weak  and  beats 
intermittently.  Pain  and  terror  combine  to  render  the  condition 
agonizing  to  the  attendant.  Convulsions  with  mild  delirium  come 
on  and  finally  death,  occasionally  from  exhaustion,  supervenes. 
This  is  the  history  of  a  severe  case,  but  ordinarily  the  symptoms  are 
less  grave,  and  severe  local  pain  accompanied  by  oedema  and  fol- 
lowed by  gangrene  are  the  results.  Some  experiments  with  the 
poison  have  been  made  on  the  lower  animals,  especially  on  rabbits 
and  guinea-pigs.  If  the  thigh  of  one  of  these  animals  be  pierced 
with  the  barb  of  one  of  these  fish  there  is  a  cry  of  pain  and  soon 
the  limb  begins  to  twitch.  The  entire  body  may  be  involved  in 
convulsive  movements,  which  resemble  those  due  to  strychnin,  inas- 
much as  they  are  intensified  by  touching  the  animal.  Respiration 
usually  becomes  difficult,  and  paralysis  of  the  posterior  extremities 


ICHTHYOTOXISMUS.  195 

often  results.  Death  may  occur  within  one  hour  after  the  infliction 
of  the  wound. 

Gressin  states  that  there  is  no  poison  gland  connected  with  the 
barbs  on  the  gill  cover  of  Trachinus  draco,  but  that  the  pocket,  in 
which  the  opercular  fin  lies,  is  lined  with  large  epithelial  cells,  which 
probably  secrete  the  poison.  This  substance  kills  small  fish,  frogs 
and  rats,  in  which  convulsions  and  fall  of  temperature  precede 
death.  One  drop  of  the  fluid  injected  subcutaneously  in  pigeons 
causes  convulsive  trembling  and  spasmodic  breathing.  While 
Gressin  found  that  the  poison  of  the  weaver-fish  at  Havre  induces 
convulsions  in  frogs,  Pohl  found  that  the  poison  of  the  same  fish 
from  the  Adriatic,  also  that  of  Trachinus  radiatus,  acts  as  an  ex- 
quisite heart  poison,  retarding  and  finally  arresting  this  organ  in 
diastole.  Its  effect  on  the  heart  is  not  altered  by  atropin,  camphor, 
caffein,  helleborein,  or  hydrastin.  Along  with  its  effects  on  the 
heart,  spontaneous  muscular  and  cutaneous  sensibility  are  impaired. 
A  similar,  though  less  active,  poison  is  found  in  the  barb  of  the 
dorsal  fin.  The  small  immovable  barbs  in  the  dorsal  fin  of  the 
Scorpcena  porcus  (hog-fish),  so  much  dreaded  by  fishermen,  are  sup- 
plied with  an  analogous  but  less  active  poison.  Neither  the  blood 
serum  nor  the  raw  flesh  of  the  Trachinus  has  poisonous  properties. 

2.  The  fish  poisoning  so  well  known  in  Japan  is  due  to  different 
species  of  the  tetrodon  (fugu).  According  to  Remy,  there  are  in 
Japan  twelve  species  of  fish,  all  belonging  to  the  genus  Tetrodon, 
whose  ovaries  are  poisonous.  In  winter  when  the  ovaries  are  atro- 
phied they  are  less  harmful ;  however,  Remy  reports  the  following 
experiments  made  with  fish  caught  during  the  winter  :  Dogs  fed  upon 
the  ovaries  or  testicles  soon  sickened,  with  salivation,  severe  and  fre- 
quent vomiting  and  convulsive  muscular  contractions.  Soon  after 
the  poison  Was  gotten  out  of  the  stomach  by  vomiting  recovery  fol- 
lowed. In  order  to  prevent  this  rapid  elimination  the  organs  were 
rubbed  up  in  a  mortar  and  the  fluid  portion  administered  subcutane- 
ously. By  this  method,  notwithstanding  the  fact  that  the  experiments 
were  made  in  winter,  death  resulted  in  less  than  two  hours.  The 
symptoms  consisted  chiefly  of  disturbances  of  the  digestive  and 
nervous  systems.  The  most  important  were  uneasiness,  salivation, 
vomiting  of  much  mucus,  severe  contractions  of  the  abdomen,  then 
paralytic  symptoms,  relaxation  of  the  sphincters,  marked  dyspnoea, 
cyanosis  and  dilatation  of  the  pupils.  Death  was  due  to  dyspnoea. 
On  section  the  salivary  glands  and  pancreas  were  found  injected  and 
hyperemic.  There  were  small  hemorrhagic  spots  in  the  stomach  and 
intestines.  The  liver  and  kidneys  were  filled  with  dark  blood  as  is 
seen  in  death  from  asphyxiation.  No  structural  changes  could  be 
found  in  the  nervous  system. 

Miura  and  Takesaki  find  that  the  ripe  ovaries  of  Tetrodon  rubripes 
contain  a  substance  which  induces  in  rabbits  acceleration  of  the  res- 


196  FOOD  POISONING. 

piratory  movements,  paralysis  of  the  skeletal  muscles,  mydriasis, 
increased  peristalsis  of  the  intestines,  and  arrest  of  the  heart. 

Takahaschi  and  Inoko  find  the  fugu  poison  resistant  to  prolonged 
boiling  and  it  behaves  like  a  basic  substance  in  its  reactions  with  the 
general  alkaloidal  reagents.  The  same  observers  claim  that  they 
have  induced  the  characteristic  symptoms  of  fugu  poisoning  by  in- 
jecting the  blood,  urine,  and  aqueous  contents  of  the  stomach  of  pa- 
tients into  the  abdominal  cavity  of  frogs. 

Tahara  reports  that  he  has  isolated  from  the  roe  of  the  tetrodon 
two  poisons.  One  of  these  is  a  crystalline  base,  to  which  he  has 
given  the  name  tetrodonin ;  while  the  other  is  a  white,  waxy  body, 
and  is  designated  as  tetrodonic  acid.  While  both  are  markedly  poi- 
sonous, the  acid  is  more  active  than  the  base.  Of  993  cases  of  fugu 
poisoning  reported  in  Tokio  from  1885  to  1892  inclusive,  680  were 
fatal — a  mortality  of  more  than  68  per  cent. 

A  disease  known  as  kakk6  was  at  one  time  very  prevalent  in 
Japan  and  other  countries  along  the  Eastern  coast  of  Asia.  Many 
theories,  some  of  which  were  quite  naturally  founded  upon  the  super- 
stitions of  that  part  of  the  world,  have  been  advanced  to  account  for 
the  etiology  of  this  disease ;  however,  with  the  opening  up  of  Japan 
to  the  civilized  world,  an  investigation  by  scientific  methods  was 
undertaken  by  foreign  physicians  and  by  the  observant  and  intelli- 
gent natives  who  had  acquired  their  medical  training  in  Europe  and 
America.  It  was  soon  ascertained  that  this  disease  was  confined  to 
the  sea  coast  districts,  and  particularly  to  the  natives,  Americans  and 
Europeans  living  in  Japan  being  almost  wholly  exempt.  With  im- 
proved transportation,  kakk4  was  found  to  extend  towards  the  in- 
terior of  Japan.  Among  the  natives,  the  most  robust  seemed  to  be 
most  prone  to  the  disease.  With  these  observations  the  following 
additional  facts  were  recognized  :  (1)  The  inhabitants  of  the  coast 
were  formerly  the  only  natives  who  partook  largely  of  sea-fish  ;  (2) 
improved  transportation  carried  these  food  products  toward  the  in- 
terior ;  (3)  the  foreigners  did  not  consume  these  fish  so  largely  as 
the  natives  did ;  (4)  among  the  natives  the  most  robust  would  quite 
naturally  eat  more  food  of  any  and  all  kinds  than  the  less  vigorous. 
The  above-mentioned  observations  led  Miura  to  define  the  disease  as 
follows :  "  Kakke  is  a  chronic  or  subacute,  seldom  an  acute,  intoxi- 
cation due  to  the  consumption  of  certain  kinds  of  fish."  He  then 
set  himself  to  solve  the  questions :  (1)  What  fish  are  the  bearers  of 
the  poison?  (2)  In  what  conditions  are  these  fish  poisonous?  In 
Tokio  the  disease  generally  appears  in  May,  reaches  its  greatest 
prevalence  in  August  and  gradually  disappears  in  September  and 
October.  This  would  indicate  that  if  the  disease  were  due  to  eating 
fish,  the  poisonous  species  must  be  those  that  are  in  demand  from 
May  to  September.  Six  species  were  found  to  be  most  abundantly, 
in  fact  almost  exclusively,  used  at  this  time  of  the  year ;  and  all  of 


ICHTHYOTOXISMUS.  197 

these  belong  to  the  family  of  Scombridse.  This  is  in  accordance 
with  the  observations  of  Gubarew,  who  has  reported  cases  of  poison- 
ing from  eating  Scombrida  saba.  However,  the  etiological  relation 
of  these  fish  to  kakk^  cannot  be  said  to  be  positively  established,  and 
it  is  true  that  in  some  parts  of  the  world  certain  species  of  the  Scom- 
bridse are  eaten  without  injurious  effects.  Nothing  definite  is  known 
about  the  nature  of  the  poison  of  these  fish,  nor  has  it  been  deter- 
mined whether  the  active  agent  is  a  physiological  product  of  certain 
glands  or  a  result  of  bacterial  activity. 

Petromyzon  fluviatilis,  which  is  not  classed  among  fish  by  modern 
zoologists,  causes,  according  to  Prochorow,  a  bloody  diarrhea,  fre- 
quently observed  in  the  Jamberg  district  of  Russia.  This  occurs 
whether  the  animal  is  eaten  raw  or  thoroughly  cooked,  and  it  is 
stated  that  if  salt  be  sprinkled  on  the  animal  while  it  is  alive  its 
skin  secretes  an  abundant  discharge  of  mucus,  and  after  this  the 
flesh  is  not  poisonous.  Bohm  and  others  have  expressed  some  doubt 
about  any  species  of  fish  being  per  se  poisonous.  They  have  been 
inclined  to  attribute  the  effect  so  frequently  observed  to  one  or  the 
other  of  the  following  causes  :  (1)  Meat  rapidly  undergoes  putre- 
factive changes  and  the  ill  effects  are  due  to  true  botulism.  (2)  The 
observed  untoward  symptoms  are  explainable  by  supposing  the  ex- 
istence of  a  marked  idiosyncrasy  in  the  consumer.  That  the  first 
supposition  is  not  true  is  shown  by  the  following  facts  :  (1)  Poison- 
ing with  perfectly  fresh  fish  occurs  not  only  in  the  tropics,  where 
decomposition  goes  on  rapidly,  but  in  the  temperate  zone  as  well, 
and  during  seasons  of  the  year  and  under  conditions  that  exclude  the 
possibility  of  the  ill  effects  being  due  to  putrefactive  changes  in  the 
meat.  (2)  Certain  species  of  tetrodon  and  other  fish  are  so  well 
known  to  be  poisonous,  even  when  perfectly  fresh,  that  their  con- 
sumption is  at  times  resorted  to,  notably  in  China  and  Japan,  for 
suicidal  purposes.  That  the  symptoms  are  not  due  to  idiosyncrasy 
in  the  consumer  is  demonstrated  by  the  effects  of  the  flesh  and  of 
the  expressed  juice  upon  the  lower  animals. 

3.  Cluppea  thrissa  and  C.  venenosa,  also  certain  species  of  Scarus, 
have  no  poisonous  glands,  nor  are  their  reproductive  organs  more 
poisonous  than  other  parts  of  the  body ;  still  the  flesh  of  these  fish  is 
always  poisonous.  According  to  Gunther,  their  harmful  properties 
are  due  to  the  medusae,  corals,  and  other  decomposing  substances 
upon  which  they  feed.  In  the  West  Indies  it  is  a  well-known  fact 
that  all  the  fish  caught  off  certain  coral  banks  are  poisonous  and  that 
every  part  of  the  animal  is  unfit  for  food.  The  symptoms  are  those 
of  a  gastroenteritis,  and  death  frequently  results. 

It  has  been  suggested  that  ichthyotoxismus  may  be  due  to  substances 
of  vegetable  origin  which  are  employed  in  some  countries,  notably 
by  savage  and  partly  civilized  peoples,  to  kill  the  fish.  That  this 
may  be  true  in  some  instances  is  possible,  but  that  this  explanation 


198  FOOD  POISONING. 

is  not  generally  applicable  is  shown  by  the  observation  that  where 
this  method  of  obtaining  fish  for  food  is  most  frequently  employed, 
no  ill  results  follow,  and  where  it  is  not  resorted  to  cases  of  fish 
poisoning  maybe  very  common.  According  to  Husemann,  Cocculus 
Indicus  has  been  employed  for  the  purpose  of  poisoning  fish.  The 
leguminous  plant  Piscidia,  of  the  West  Indies,  owes  its  name  to  this 
use  of  its  bark.  In  the  Dutch  East  Indies  the  cortex  of  the  root  of 
Derris  elliptica  and  the  seed  of  Pachyrrhinus  angulatus  are  employed 
for  this  purpose.  Both  of  these,  according  to  Greshof,  contain  a 
non-nitrogenous  substance  which  is  highly  poisonous  to  fish,  and  rela- 
tively harmless  to  other  animals.  An  extract  of  the  derris  root, 
which,  in  Borneo,  is  also  used  as  an  arrow  poison,  kills  fish  when 
mixed  with  water  in  the  proportion  of  1 :  25,000,  and  the  active  prin- 
ciple in  a  dilution  of  1 : 5,000,000.  Greshof  has  isolated  both  of 
these  poisons  and  named  them  derrid  and  pachyrrhizid.  A  legumen, 
Tephrosia  ichthyonecea,  from  West  Africa,  also  yields  a  non-ni- 
trogenous poison,  but  this  affects  other  animals  as  well  as  fish.  The 
fish  poison  of  Java,  from  the  seed  of  Milletia  atropurpurea,  contains 
saponin,  and  that  of  Ceylon,  from  Hydrocarpos  inebrians,  owes  its 
effects  to  hydrocyanic  acid.  E-obinia  nicon  of  tropical  America  is 
used  by  the  savage  tribes  for  the  purpose  of  benumbing  fish  and  this 
plant  has  been  found  to  contain  a  snow-white  crystalline  substance, 
freely  soluble  in  alcohol,  wholly  insoluble  in  water.  Water,  to 
which  an  alcoholic  solution  of  this  poison  has  been  added  in  the  pro- 
portion of  1  : 1,000,000,  killed  fish.  Other  fish  poisons  of  the  West 
Indies  are  Jacquinia  armillaris,  which,  on  account  of  the  fact  that  its 
dried  fruit  is  used  for  bracelets  is  known  as  hois  hracelet,  and  Ser- 
jania  letalis,  from  which  the  poisonous  honey  of  a  certain  wasp  is 
prepared,  the  toxic  action  of  which  St.  Hilaire  tested  upon  himself. 
This  honey,  even  in  small  quantity,  is  said  to  produce  a  mild  intoxi- 
cation. This  will  remind  the  classical  student  of  the  poisonous 
honey  connected  with  the  retreat  of  the  ten  thousand  Greek  sol- 
diers under  Xenophon,  which  occurred  four  hundred  years  before 
our  era. 

4.  We  include  in  this  class  the  cases  that  Blanchard  would  de- 
scribe under  botulism,  inasmuch  as  the  poisoning  is  due  to  putrefac- 
tive changes.  According  to  Anrep,  there  are  in  poisonous  fish  two 
active  ptomains.  One  of  these  is  extracted  from  alkaline  solution 
with  ether,  chloroform  and  benzin.  It  is  amorphous  and  insoluble 
in  water,  but  forms  easily  soluble  salts  of  great  toxicity,  so  that  one- 
fourth  mg.  of  the  hydrochlorid  induces  poisonous  effects  in  dogs,  and 
one-half  mg.  kills  rabbits.  This  ptomain  may  be  preserved  quite 
indefinitely  in  the  dry  state  or  dissolved  in  ether,  but  is  speedily  de- 
stroyed by  strong  alkalis  and  acids.  Dissolved  in  phosphoric  acid 
and  evaporated  it  gives  a  red  coloration,  rapidly  passing  into  a  dirty 
green.     Jakolew  isolated  a  similar  alkaloid  from  poisonous  sturgeon 


ICHTHYOTOXISMUS.  199 

in  1889,  but  it  differed  from  that  of  Anrep  in  the  fact  that  the 
former  gave  precipitates  with  platinum  chlorid  and  tannin,  while  the 
latter  did  not.  Anrep's  second  ptomain  is  an  oily  substance,  less 
poisonous  than  the  solid.  Both  these  substances  have  a  paralyzing 
action  on  frogs,  dogs  and  rabbits,  arresting  respiration  and  the  ac- 
tion of  the  heart.  In  cats,  they  cause  clonic  convulsions.  The 
heart's  action  is  retarded,  and  just  before  death  respiration  is  accel- 
erated. The  more  poisonous  of  these  alkaloids,  for  which  the  name 
halichthytoxin  has  been  suggested,  produces  mydriasis  on  applica- 
tion to  the  eye. 

Vaughan  reports  the  following  case  of  ichthyotoxismus  exanthe- 
maticus  :  K.,  a  vigorous  man  of  thirty-four  years,  ate  freely  of  canned 
salmon,  while  others  at  the  table  with  him  remarked  that  the  taste 
of  the  fish  was  peculiar  and  refrained  from  eating  it.  Twelve  hours 
later  K.  began  to  suffer  from  nausea,  vomiting,  and  a  griping  pain 
in  the  abdomen ;  and  six  hours  later  he  was  found  vomiting  small 
quantities  of  mucus,  colored  with  bile,  at  frequent  intervals.  The 
bowels  had  not  moved  and  the  griping  pain  continued,  while  he  was 
covered  with  a  scarlatinous  rash  from  head  to  foot.  The  pulse  was 
140,  the  temperature  102°  F.,  and  respiration  shallow  and  irregular. 
The  stomach  and  large  intestines  were  washed  out  thoroughly,  and 
ten  grains  of  calomel,  soon  followed  by  twelve  ounces  of  solution  of 
magnesium  citrate,  for  the  purpose  of  cleansing  the  small  intestines, 
were  administered.  The  next  day  the  rash  disappeared,  but  the 
temperature  remained  above  the  normal  for  four  or  five  days,  and  it 
was  not  until  a  week  later  that  this  man  was  able  to  leave  his  house. 
The  absence  of  inorganic  poisons  in  the  salmon  was  demonstrated 
and  it  was  found  that  subcutaneous  injection  of  twenty  drops  of  the 
fluid  contained  in  the  can  caused  evident  illness  and  suffering  in  a 
white  rat.  The  only  microorganism  that  could  be  found,  either  by 
direct  microscopical  examination  or  by  the  preparation  of  plate 
cultures,  was  a  micrococcus,  and  this  was  present  in  the  salmon 
in  great  numbers.  This  germ  grew  fairly  well  in  beef-tea,  but 
the  injection  of  five  c.c.  of  beef-tea  cultures  of  different  ages 
failed  to  affect  white  rats,  kittens  or  rabbits.  However,  this  micro- 
coccus, when  grown  for  twenty  days  in  a  sterilized  egg  produced  a 
most  potent  poison.  The  white  of  the  egg  became  thin,'  watery, 
markedly  alkaline,  and  ten  drops  sufficed  to  kill  white  rats.  It  is 
supposed  that  this  micro5rganism  produced  toxins  only  when  grown 
under  anaerobic  conditions.  The  same  observer  saw  two  similar 
cases  also  due  to  eating  canned  salmon,  and  in  one  of  these,  the  at- 
tending physician  having  arrested  the  vomiting  and  purging  by  the 
hypodermatic  administration  of  morphin,  death  resulted.  This 
illustrates  the  danger  of  interfering  with  nature's  efforts  to  remove 
the  poison  which,  instead  of  being  arrested,  should  have  been  assisted 
by  the  use  of  the  stomach  tube  and  by  irrigation  of  the  colon. 


200  FOOD  POISONING. 

Potain  saw  a  man  sufFering  from  vomiting,  vertigo,  ringing  in  the 
ears,  and  pain  in  the  joints,  due  to  eating  lobster. 

Griffiths  found  in  sardines  that  had  undergone  putrefactive 
changes  a  base  to  which  he  has  given  the  name  sardinin ;  thxs  pto- 
main,  together  with  others  reported  by  the  same  investigator,  is  de- 
scribed in  Chapter  XIV. 

5.  In  Russia  many  instances  of  fish  poisoning  are  due  to  the  fact 
that  the  fish  are  diseased  and  the  disease  is  transmitted  to  man  in 
his  food.  Instances  of  this  kind  of  fish  poisoning  are  well  known 
in  Germany  also,  and  here  they  are  generally  due  to  eating  diseased 
barbels.  The  symptoms  are  identical  with  those  of  cholera  nostras, 
and  the  disease  is  known  as  "  barbencholera."  The  poison,  the 
nature  of  which  is  yet  unknown,  evidently  irritates  the  mucous  mem- 
brane of  the  stomach  and  intestines.  This  form  of  fish  poisoning  is 
sometimes  called  ichthyotoxismus  gastricus. 

Schmidt  concludes  his  studies  on  poisonous  fish  in  Russia  with 
the  following  statements  :  (1)  Poisoning  with  fish  is  not  due  to 
putrefaction.  (2)  Fish  poisoning  (in  Russia)  is  always  due  to  some 
member  of  the  sturgeon  tribe.  (3)  The  genesis  of  fish  poisoning 
has  no  relation  to  the  method  of  catching  the  fish,  the  use  of  salt,  or 
imperfections  in  the  method  of  preserving  them.  (4)  The  poisonous 
substance  is  not  distributed  throughout  the  animal,  but  is  confined  to 
certain  parts.  (5)  The  poisonous  portion  cannot  be  distinguished 
from  the  non-poisonous,  either  macroscopically  or  microscopically. 
(6)  The  thoroughly  cooked  meat  is  never  poisonous.  (7)  The  fish 
poison  is  an  animal  alkaloid,  produced  most  probably  by  bacteria 
that  cause  an  infectious  disease  in  the  fish. 

Arustamow  has  studied  eleven  cases  of  fish  poisoning  in  which 
five  terminated  fatally.  In  the  fish,  and  in  the  liver,  kidneys  and 
spleen  of  the  persons,  germs,  resembling  but  not  identical  with  the 
typhoid  bacillus,  were  found.  The  most  noteworthy  symptoms  were 
general  weakness,  dull  pain  in  the  abdomen,  dyspnoea,  mydriasis,  ver- 
tigo, and  dryness  of  the  mouth.  This  author  also  concludes  that  the 
ill  effects  are  due  to  bacteria  which  are  pathogenic  to  the  fish  and  in 
the  cases  observed  by  him  the  meat  was  eaten  raw. 

Sieber  found  that  the  fish  in  an  aquarium,  from  which  some  had 
been  taken  to  supply  a  table  and  had  proved  poisonous,  were  sick 
and  that  as  many  as  thirty  died  within  the  next  two  days.  From 
the  dead  and  sick  fish  Sieber  obtained  by  anaerobic  methods  a  highly 
toxicogenic  germ  to  which  she  has  given  the  name  Bacillus  piscicidus 
agilis.  This  germ  consists  of  highly  motile  short  rods,  and  old 
cultures  show  spore  formation.  The  bacilli  are  easily  colored  with 
Ziehl's  solution  and  on  gelatin  and  agar  plates  the  colonies  are  gran- 
ular, gray,  or  yellow.  This  organism  liquefies  gelatin  and  produces 
carbonic  acid  gas  and  small  quantities  of  methyl  mercaptan.  It  is 
pathogenic  to  fish,  frogs,  mice,  rabbits,  dogs  and  guinea-pigs,  and 


KBEOTOXISMUS.  201 

from  the  muscles  of  these  animals  the  germ  may  be  recovered  in 
pure  culture.  After  filtration  through  porcelain  the  sterile  cultures 
are  quite  as  poisonous  as  before  sterilization.  Heat  does  not  de- 
stroy the  toxin  and  at  least  one  poisonous  substance  may  be 
obtained  by  distillation.  Filtered  cultures  give  an  intense  red 
coloration  with  ferric  chlorid.  Sieber  has  obtained  from  growths  of 
this  bacillus  cadaverin  and  other  known  ptomains,  but  there  are  at 
least  two  undetermined  bases  present  and  one  of  these  suffices  to  kill 
frogs  in  doses  of  3.5  mg.  The  symptoms  induced  in  animals  by  the 
use  of  sterilized  cultures  consist  of  shortness  of  breath  and  unrest, 
followed  by  apathy  and  paralysis. 

Kreotoxismus. — It  has  long  been  known  that  the  flesh  of  animals 
dead  from  certain  diseases  or  slaughtered  while  suffering  from  these 
diseases,  is  not  a  safe  food  for  man.  The  Mosaic  law  forbade  the 
eating  of  the  flesh  of  animals  dead  from  disease  :  "  Ye  shall  not  eat  of 
anything  that  dieth  of  itself ;  thou  shalt  give  it  unto  the  stranger  that 
is  in  thy  gates,  that  he  may  eat  it ;  or  thou  mayst  sell  it  unto  an  alien, 
for  thou  art  a  holy  people  unto  the  Lord  thy  God.  Thou  shalt  not 
seethe  a  kid  in  his  mother's  milk."  (Deuteronomy,  XIV,  21.)  The 
first  part  of  this  command  is  certainly  wise  counsel,  but  the  feeding 
of  a  visitor  with  such  food  would  not  be  now  regarded  as  in  accord 
with  the  rules  of  hospitable  entertainment,  and  the  sale  of  it  even  to 
an  alien  should  not  be  permitted  by  the  law  of  any  country.  The 
most  common  diseases  that  may  be  transmitted  from  the  lower  ani- 
mals to  man  by  the  consumption  of  the  flesh  or  milk  of  the  former 
as  food  by  the  latter,  are  tuberculosis,  anthrax,  symptomatic  anthrax, 
pleuro-pneumonia,  puerperal  fever,  glanders,  various  septicemias, 
trichinosis,  mucous  diarrhcea,  and  actinomycosis.  However,  it  does 
not  come  within  the  scope  of  this  book  to  discuss  the  transmission 
of  these  diseases  from  the  lower  animals  to  man,  and  we  shall  limit 
the  subject  of  kreotoxismus  to  those  untoward  effects  which  arise 
from  the  eating  of  flesh  infected  with  non-specific,  toxicogenic  bac- 
teria. 

Sausage  poisoning,  sometimes  designated  as  botulism  or  botulis- 
mus,  and  sometimes  known  as  allantiasis,  has  long  been  recognized 
as  a  cause  of  sickness  and  death  and  its  causation  has  been  a  subject 
of  theory  and  investigation  for  one  hundred  years  or  longer.  It  is 
probable  that  some  of  the  earlier  epidemics  attributed  to  botulism 
were  in  fact  due  to  trichiniasis,  and  it  was  not  until  the  discovery  of 
the  parasite  to  which  the  latter  condition  is  due  that  differentiation 
was  possible.  A  large  proportion  of  the  cases  of  sausage  poisoning 
have  occurred  in  Wiirtemberg  and  the  immediately  adjacent  portions 
of  Baden.  This  fact  has  been  correctly  ascribed  to  the  methods 
there  practised  of  preparing  and  curing  sausage.  It  is  said  to  be 
common  for  people  to  use  the  blood  of  the  sheep,  ox  and  goat  in  the 


202  FOOD  POISONING. 

preparation  of  this  article  of  diet.  Moreover,  the  blood  is  kept  for 
days  sometimes  in  wooden  boxes  and  at  a  high  temperature  before 
it  is  used.  In  these  cases  it  is  altogether  likely  that  putrefaction 
progresses  to  the  poisonous  stage  before  the  process  of  curing  is  be- 
gun. A  kind  of  sausage  known  as  "  blunzen  "  is  made  by  filling 
the  stomachs  of  hogs  with  the  meat.  In  curing,  the  interior  of  this 
great  mass  is  not  acted  upon  and  putrefaction  sets  in.  The  curing 
is  usually  done  by  hanging  the  sausage  in  the  chimney  and  at  night 
the  fire  goes  out  and  the  meat  freezes.  The  interior  of  the  mass  is 
generally  the  most  poisonous  part  and  in  many  instances  those  who 
have  eaten  of  the  outer  portion  are  often  unharmed,  while  those  who 
have  eaten  of  the  interior  of  the  same  sausage  have  been  seriously 
affected.  This  method  of  preparing  sausage  in  Wiirtemberg  is  not 
now  so  generally  employed  and  poisoning  from  this  article  of  food  is 
not  so  common  as  formerly. 

Many  German  writers  state  that  when  a  poisonous  sausage  is  cut, 
the  putrid  portion  has  a  dirty,  grayish-green  color,  and  a  soft,  smeary 
consistency.  A  disagreeable  odor,  resembling  that  of  putrid  cheese, 
is  perceptible,  while  the  taste  is  unpleasant  and  sometimes  a  smarting 
of  the  mouth  and  throat  is  produced.  Post-mortem  examination 
shows  no  characteristic  lesions.  It  is  generally  stated  that  putrefac- 
tion sets  in  very  tardily,  but  Miiller  shows  that  no  reliance  can  be 
placed  upon  this  point,  and  states  that  out  of  forty-eight  recorded  au- 
topsies, it  was  especially  stated  in  eleven  that  putrefaction  rapidly  de- 
veloped. In  some  instances  there  has  been  noticed  hyperemia  of  the 
stomach  and  intestinal  canal,  but  this  is  by  no  means  constant.  The 
liver  and  brain  have  been  reported  as  congested,  but  this  would  re- 
sult from  failure  of  the  heart,  and  would,  by  no  means,  be  character- 
istic of  poisoning  with  sausage. 

Von  Faber,  in  1821,  observed  sixteen  persons  who  were  made 
sick  by  eating  fresh  unsmoked  sausage  made  from  the  flesh  of  a  pig 
which  had  suffered  from  an  abscess  on  the  neck.  Five  of  the 
patients  died.  The  symptoms  were  as  follows  :  There  was  con- 
striction of  the  throat,  difficulty  in  swallowing,  retching,  vomiting, 
colic-like  pains,  vertigo,  hoarseness,  dimness  of  vision  and  headache. 
Later  on  in  severer  cases  there  was  complete  exhaustion,  and,  finally, 
paralysis.  The  eyeballs  were  retracted,  the  pupils  were  sometimes 
dilated,  then  contracted,  they  did  not  respond  to  light,  there  was 
paralysis  of  the  upper  lids.  The  tonsils  were  swollen,  but  not  as  in 
tonsillitis.  Liquids  which  were  not  irritating  could  be  carried  as  far 
as  the  esophagus,  when  they  were  ejected  from  the  mouth  and  nose 
with  coughing.  Solid  foods  could  not  be  swallowed.  On  the  back  of 
the  tongue  and  in  the  pharynx  there  was  observed  a  puriform  exudate. 
Obstinate  constipation  existed  in  all,  while  the  sphincter  ani  was 
paralyzed.  Breathing  was  easy,  but  all  had  a  croupous  cough.  The 
skin  was  dry  and  there   was  incontinence  of  urine.     There  was  no 


KREOTOXISMUS.  203 

delirium  and  the  mind  remained  clear  to  the  last.  At  autopsy  the 
skin  was  found  to  be  rough  and  the  abdomen  retracted.  The  large 
vessels  in  the  upper  part  of  the  stomach  were  filled  with  black  blood 
and  the  content  of  the  stomach  consisted  of  a  reddish-brown,  semi- 
fluid substance,  which  gave  off  a  repugnant  acid  odor.  In  one  case 
the  omentum  was  found  greatly  congested.  The  large  intestine  was 
pale  and  the  right  ventricle  of  the  heart  was  filled  with  dark  fluid 
blood. 

Schiiz  cites  thirteen  cases  of  poisoning  from  liver  sausage  in  which 
the  symptoms  differed  from  the  foregoing  in  the  following  respects : 
(1)  In  only  one  out  of  the  thirteen  was  there  constipation.  All  the 
others  had  numerous  typhoid-like  stools.  (2)  Symptoms  involving 
the  sense  of  sight  were  present  in  only  three ;  in  all  the  pupils  were 
unchanged.  (3)  The  croupous  cough  was  wholly  wanting;  though 
in  many  there  was  complete  loss  of  voice.  Difficulty  of  swallowing 
was  complained  of  by  only  one.  (4)  Delirium  was  marked  in  all; 
and  in  one  the  disturbance  of  the  mental  faculties  was  prominent  for 
several  weeks.  (5)  There  were  no  deaths.  (6)  The  time  between 
eating  of  the  sausage  and  the  appearance  of  the  symptoms  varied 
from  eighteen  to  twenty-four  hours  and  the  duration  of  sickness  from 
one  to  four  weeks ;  though  in  one  case  complete  recovery  did  not 
occur  until  after  two  and  one-half  months.  The  sausages  were  not 
smoked  and  all  observed  a  garlic  odor,  though  no  garlic  had  been 
added  to  the  meat. 

Tripe  reports  sixty-four  cases,  in  which  the  stools  were  frequent, 
watery,  and  of  offensive  odor ;  in  some  there  was  delirium.  In  the 
one  fatal  case  the  hands  and  face  were  cold  and  swollen ;  the  pulse, 
rapid  and  weak  ;  the  pupils,  contracted  but  responsive  to  light ;  and 
the  small  intestine,  inflamed. 

Hedinger  reports  two  cases  with  the  usual  symptoms,  but  during 
recovery  dilatation  of  the  pupils  was  followed  by  contraction.  Birds 
ate  of  this  sausage  and  were  not  affected.  In  Roser's  cases,  section 
showed  abscess  of  the  tonsils,  a  dark,  bluish  appearance  of  the 
mucous  membrane  of  the  pharynx,  larynx,  and  bronchial  tubes,  dark 
redness  of  the  fundus  of  the  stomach,  and  circumscribed  gray,  red 
and  black  spots  on  the  mucous  membrane  of  the  intestine  ;  the  liver 
was  brittle  and  the  spleen  solid. 

Many  theories  concerning  the  nature  of  the  active  principle  of 
poisonous  sausage  have  been  advanced.  It  was  once  believed  to 
consist  of  pyroligneous  acid,  supposed  to  be  absorbed  by  the  meat 
from  the  smoke  used  in  curing,  but  it  was  found  that  unsmoked 
sausage  also  might  be  poisonous.  Emmert  believed  the  active  agent 
to  be  hydrocyanic  acid,  and  Jager's  theory  supposed  the  presence  of 
picric  acid  ;  but  these  acids  are  not  found  in  poisonous  sausage  and 
their  toxicological  effects  are  wholly  unlike  those  observed  in  sausage 
poisoning.     Kerner  believed  the  poison  to  consist  of  either  caseic  or 


204  FOOD  POISONING. 

sebacic  acids,  or  both,  while  Buchner  named  it  acidum  botulinicum  ; 
but  the  acids  of  the  former  proved  to  be  inert,  and  that  of  the  latter 
to  have  no  existence.  Schlossberger  suggested  that  the  poisonous 
substance  is  most  probably  basic  in  character,  and  he  found  an 
ammoniacal  base  which  could  not  be  found  in  good  sausage,  and 
which  did  not  correspond  to  any  known  amid,  imid,  or  nitryl  base ; 
however,  this  substance  has  not  been  obtained  by  anyone  else,  nor  has 
it  been  demonstrated  to  be  poisonous.  Liebig,  Duflas,  Hirsch,  and 
Simon  believed  in  the  presence  of  a  poisonous  ferment,  and  Van  den 
Corput  described  sarcina  botulina,  which  he  believed  to  be  the  active 
agent.  Miiller,  Hoppe-Seyler,  and  others  found  various  microor- 
ganisms, and  Virchow,  Eichenberg,  and  others  examined  micro- 
scopically the  blood  of  persons  poisoned  "with  sausage.  Ehrenberg 
attempted  to  isolate  the  poisonous  body  by  employing  Brieger's 
method,  but  obtained  only  inert  substances. 

Gaffky  and  Paak  made  a  study  of  sausage  consisting  of  horse- 
flesh and  liver  which  poisoned  a  large  number  of  people,  one  of 
whom  died.  In  the  majority,  the  symptoms  came  on  with  a  chill 
within  six  hours,  and  in  one  instance  within  half  an  hour.  The 
most  prominent  symptoms  were  headache,  loss  of  appetite,  pain  in 
the  bowels,  vomiting  and  purging.  From  the  sausage  these  investi- 
gators obtained  a  short  bacillus,  which,  when  given  by  the  mouth, 
subcutaneously  or  intravenously,  produced  the  above-mentioned 
symptoms,  with  a  fatal  termination  in  rabbits,  guinea-pigs,  mice  and 
apes  ;  they  were  unable  to  isolate  the  chemical  poison.  This  bacillus 
probably  belongs  to  the  colon  group. 

In  June,  1880,  a  large  number  of  persons  were  poisoned  at  Well- 
beck,  England,  by  eating  ham  in  which  Klein  found  a  bacillus,  in- 
oculations with  which  were  followed  by  the  development  of  pneu- 
monia ;  while  the  affected  persons  were  described  by  Ballard,  who 
investigated  the  outbreak,  as  suffering  from  "diarrhoeal  illness." 
Ballard  has  reported  the  following  additional  instances :  In  the 
"  Chester  case,"  the  man  ate  of  some  so-called  American  sausage, 
which  consisted  mostly  of  pork.  Gastroenteric  symptoms  with 
great  prostration  resulted,  and  in  a  few  days  the  man  died,  apparently 
from  pneumonia.  No  post-mortem  examination  was  permitted,  but 
the  meat  killed  the  animals  fed  with  it,  and  in  these,  section  showed 
hemorrhage  in  the  stomach,  congestion  of  the  lungs,  and  hyperemia 
of  the  medullary  portion  of  the  kidneys.  In  the  "  Oldham  case," 
members  of  two  families  partook  of  a  newly  opened  can  of  pigs' 
tongues.  Nausea,  vomiting  and  diarrhoea  occurred  in  all  but  one, 
and  he  fell  into  a  comatose  condition,  which  was  not  relieved  until 
a  purgative  was  administered  and  acted.  In  the  "  Bishop  case," 
members  of  three  families  ate  of  ribs  of  beef.  The  meat  was 
cooked  on  Saturday  and  it  was  more  poisonous  on  Monday  than  on 
the  preceding  day.     This  meat  evidently  became  infected  after  it 


KBEOTOXISMUS.  205 

was  cooked,  inasmuch  as  other  portions  of  the  same  carcass  caused 
no  ill  effects.  In  the  "  Whitechurch  case,"  brawn,  consisting  of  a 
gelatin  made  from  pig's  head,  was  eaten  cold.  Members  of  ten  fam- 
ilies residing  in  different  parts  of  the  town  were  affected.  In  the 
"  Whitechurch-pork  case,"  the  meat  eaten  immediately  after  it  was 
cooked  had  no  ill  effect,  while  portions  eaten  cold  the  next  day- 
killed  two  persons.  In  the  "  Wolverhampton-tin-salmon  case,"  three 
adults  ate,  and  two  children  merely  tasted,  the  contents  of  a 
"  blown "  can.  The  one  who  ate  the  most  was  attacked  about  ten 
hours  after  eating,  and  died  in  three  days ;  one  who  ate  less  became 
ill  in  about  twelve  hours,  and  died  in  five  days  ;  while  the  adult  who 
partook  most  sparingly  began  to  feel  the  ill  effects  in  about  fourteen 
hours,  and  finally  recovered.  In  the  children,  the  symptoms  were 
slight  and  transient.  Klein  found  in  both  fatal  cases  necrosis  of  the 
superficial  layers  of  the  mucous  membrane  of  the  stomach,  fatty  de- 
generation of  the  liver  as  in  acute  phosphorus  poisoning,  and  inflam- 
mation of  the  kidneys.  Mice  fed  upon  the  salmon  died,  exhibiting 
lesions  similar  to  those  observed  in  the  men  ;  no  germ  could  be  found 
in  the  blood  of  the  mice.  In  the  "  Carlisle  (A)  case,"  twenty-four 
persons  partook  of  a  cold  ham,  which  had  been  prepared  the  pre- 
vious day  and  kept  in  a  cellar  in  which  milk  and  meat  "  were  known 
to  go  bad."  Two  persons  died.  The  only  germ  which  could  be 
found  was  a  micrococcus,  which  was  harmless,  when  fed  by  mouth 
to  mice,  cats  and  dogs.  In  the  "  Ironbridge  case,"  twelve  persons 
out  of  fifteen  in  a  household  ate  at  midday  of  veal  pie  which  had 
been  made  the  day  before  and  warmed  over.  Mice  were  fed  upon 
the  pie  and  upon  portions  of  the  veal  from  which  the  pie  had  been 
made  ;  those  fed  on  the  veal  showed  no  ill  effects,  while  some  of  those 
fed  on  the  pie  died.  In  the  pie  several  different  species  of  microor- 
ganisms were  found,  and  with  one  bacterium  the  following  interest- 
ing observations  were  made  :  Cultures  did  not  grow  well  at  a  tem- 
perature over  32°  and  at  above  36°  no  growth  took  place.  All 
cultures  after  some  days  growth  possessed  a  most  exquisite  and 
delicate  aroma,  no  trace  of  putridity  being  perceptible. 

As  might  be  expected  from  the  fact  that  the  bacterium  did  not 
grow  at  blood  heat,  subcutaneous  inoculations  into  mice  produced  no 
results  ;  but  when  mice  were  fed  with  the  contents  of  a  culture  tube, 
they  fell  ill  and  died,  but  no  bacterium  could  be  found  in  any  of  the 
viscera.  The  obvious  inference  from  this  is  that  the  cultures  of  this 
microorganism  contained  a  substance  which,  when  introduced  into 
the  stomach,  produced  illness  and  death,  in  the  latter  event  severe 
gastro-enteritis  being  a  conspicuous  feature.  Since  the  organism  in 
itself  is  harmless  when  inoculated,  not  being  capable  of  growth  and 
multiplication  at  the  temperature  of  the  animal  body,  it  follows  that 
the  substance  which  caused  the  poisoning  was  non-organized  and 
produced  by  the  bacterium.     In  the  "  Retford  case,"  eighty  persons 


206  FOOD  POISONING. 

in  twenty-two  families  were  made  ill,  one  fatally,  by  eating  pork 
pie.  The  meat  was  cooked  on  November  lOtli  and  was  eaten  from 
the  11th  to  the  14th.  With  the  exception  of  one  family,  none  of 
those  who  ate  of  the  pie  on  the  11th  were  made  ill,  and  none  of 
those  who  ate  of  it  after  the  14th.  The  harmful  germ  was  a  short 
bacillus,  cultures  of  which  made  mice  sick,  killing  some ;  but 
growths  more  than  ten  days  old  were  without  effect.  In  the  "  Car- 
lisle (B)  case,"  a  pork  pie  was  made  November  1st  and  eaten  by 
some  twenty-five  persons  during  the  following  ten  days.  Mice  fed 
upon  the  meat  developed  on  the  second  or  third  day  a  bloody  diar- 
rhoea, and  died.  The  small  intestine  was  filled  with  bloody  mucus. 
The  lungs  were  congested,  and  in  the  animals  which  lived  the  longest 
there  was  hepatization,  chiefly  in  the  upper  lobe ;  the  liver  and 
spleen  were  also  congested.  In  the  "  Portsmouth  case,"  two  bacilli 
— one  motile,  the  other  not — were  found  in  the  food.  When  first 
received,  the  meat  poisoned  mice  fed  upon  it,  but  after  standing  and 
becoming  offensive  in  odor,  it  failed  to  do  so.  Cultures  of  the  non- 
motile  bacillus  had  a  pleasant  aroma,  and  were  poisonous  ;  while 
those  of  the  motile  germ  were  offensive  and  harmless.  In  the  ani- 
mals killed  by  the  culture  the  lungs,  kidneys  and  spleen  were  dark 
and  the  small  intestines  were  relaxed  and  filled  with  mucus.  In 
only  one  of  the  mice  could  the  bacillus  be  found,  and  Klein  states  that 
this  germ  is  not  pathogenic  but  that  its  cultures  contain  a  toxin.  In 
the  "  Middlesborough  Pneumonia  Epidemic,"  Ballard  attributed  490 
deaths  to  infected  bacon,  and  he  believed  that  the  disease,  developed 
in  a  person  who  had  eaten  of  the  infected  food,  was  transmissible 
from  the  sick  to  those  who  had  not  partaken  of  the  bacon.  This 
has  been  observed  in  other  epidemics  of  kreotoxismus  and  is  due 
to  infection  with  microorganisms  first  obtained  in  the  food  and 
then  transmissible,  probably  through  the  sputum,  to  others.  In  the 
lungs,  Klein  discovered  a  short  bacillus,  which  he  called  "  bacillus 
pneumoniae,"  differing  altogether  from  the  bacillus  of  Friedliinder 
and  from  the  diplococcus  of  Frankel,  neither  of  which  was  present. 
Of  twenty  samples  of  bacon  forwarded  from  the  infected  districts,  four- 
teen were  distinctly  poisonous  to  rodents  fed  with  it ;  in  two  in- 
stances, there  was  some  doubt,  and  only  four  proved  not  to  be  poison- 
ous. In  the  dead  animals,  lesions  similar  to  those  observed  in 
persons  who  died  of  the  disease,  in  the  infected  districts,  were  found. 
Similar  results  followed  inoculation  of  the  juice  expressed  from  the 
lungs  and  of  pure  cultures  of  the  bacillus,  and  in  all  instances  the 
microorganism  was  recoverable.  During  the  progress  of  Klein's 
investigations  an  epidemic  of  pneumonia  occurred  among  the  animals 
(mice,  guinea-pigs,  and  monkeys)  kept  in  the  building  where  his  ex- 
periments were  carried  on,  the  bacillus  pneumoniae  being  found  after 
death  and  sometimes  in  the  heart's  blood.  On  reexamination  after 
the  lapse  of  three  months,  of  samples  of  the  bacon  that  had  previously 


KREOTOXISMUS.  207 

produced  illness  and  death,  they  were  found  to  have  lost  their  power 
of  infecting  animals,  and  no  growth  of  the  bacillus  was  obtainable. 
In  meat  which  had  poisoned  a  large  number  of  persons,  Gaertner 
found  his  bacillus  enteritidis.  The  meat  was  from  a  cow  that  had  a 
severe  diarrhoea  for  two  days  before  she  was  killed.  The  twelve 
persons  who  ate  of  the  flesh  raw,  all  became  sick ;  while  of  those 
who  ate  of  the  cooked  food  a  large  per  cent,  were  also  affected.  In 
the  meat,  and  in  the  spleen  of  a  person  who  died  from  the  effects  of 
the  poison,  Gaertner  found  the  bacillus  which  proved  fatal  to  animals. 
Good  beef,  inoculated  with  this  germ  and  kept  for  some  hours,  killed 
rabbits,  guinea-pigs  and  mice.  The  skin  of  the  people  who  were 
poisoned  and  recovered  peeled  off.  The  period  of  incubation  varied 
from  two  to  thirty  hours.  Even  the  boiled  bouillon  cultures  of  this 
germ  are  highly  poisonous,  showing  that  the  toxic  properties  are  not 
destroyed  by  cooking  the  meat.  Fischer  reports  the  following:  A 
cow,  that  had  recently  calved,  had  been  sick  for  some  eight  days,  and 
on  account  of  this  illness  she  was  killed.  The  animal  was  slaughtered 
on  Friday,  and  on  the  following  Sunday  at  noon  nineteen  persons 
ate  of  the  meat.  The  prominent  symptoms  were  vomiting  and  violent 
purging,  appearing  a  few  hours  after  the  meal.  Vertigo,  loss  of  con- 
sciousness, and  exfoliation  of  the  epidermis  during  recovery,  all  of 
which  were  observed  by  Gaertner  in  some  of  his  cases,  were  not 
present  in  any  case  reported  by  Fischer.  Notwithstanding  these 
differences,  a  study  of  the  bacillus  led  to  the  conclusion  that  it  is 
identical  with  the  bacillus  enteritidis.  By  concentrating  a  filtered 
culture  and  precipitating  with  absolute  alcohol,  the  crude  toxin  was 
obtained.  It  gave  the  general  reactions  for  peptons,  and  boiling 
for  one  and  one-half  hours  did  not  perceptibly  weaken  its  toxic  prop- 
erties. Lubarsch  reported  the  death  of  a  child  two  days  old  from  a 
septic  pneumonia  caused  by  the  bacillus  enteritidis.  Section  showed 
pleuritis  and  pneumonia  of  the  left  lower  lobe,  bilateral  purulent 
bronchitis,  atalectasis  of  the  right  lung,  parenchymatous  cloudiness 
of  the  kidneys,  fatty  infiltration  and  engorgement  of  the  liver,  slightly 
enlarged  spleen,  uric  acid  infarction  of  the  kidneys,  and  icterus  neo- 
natorum. All  other  pathological  conditions  were  supposed  to  be 
consequent  upon  the  septic  pneumonia.  White  rats  and  chickens 
proved  to  be  wholly  immune,  while  guinea-pigs,  rabbits,  and  mice 
were  susceptible  to  the  bacillus  found  in  the  tissues  of  the  child. 
These  susceptible  animals  were  killed  within  from  sixteen  to  twenty- 
four  hours  by  intraperitoneal  inoculation  and  in  from  two  to  four  days 
by  subcutaneous  injections.  In  all  cases,  section  showed  marked 
congestion  of  the  intestines,  swelling  of  the  follicles,  and  in  some 
instances  slight  erosions  of  the  mucous  membrane.  After  intraperi- 
toneal inoculation,  sero-fibrinous  or  hemorrhagic  peritonitis  de- 
veloped. After  subcutaneous  inoculation  in  rabbits,  sometimes  in 
guinea-pigs,  there  was  a  sero-fibrinous  pleuritis,  with  compression  of 


208  FOOD  POISONING. 

the  lungs,  and  in  one  instance  a  circumscribed  pneumonia.  Sterilized 
cultures  in  larger  quantities  produced  the  same  eflfects  as  the  unster- 
ilized.  The  symptoms  and  anatomical  changes  induced  by  this  germ 
agree  with  those  observed  in  Winkel's  disease,  in  the  rapidly  fatal 
progress,  cyanosis,  icterus,  rapid  respiration,  tendency  to  hemorrhage 
and  fatty  degeneration.  The  most  essential  difference  lies  in  the  fact 
that  hemoglobinuria  is  a  prominent  symptom  of  Winkel's  disease, 
while  it  does  not  occur  after  inoculation  with  the  bacillus  enteritidis. 

In  August,  1887,  256  soldiers  and  thirty-six  citizens  at  Middle- 
burg,  Holland,  were  taken  sick  after  eating  meat  from  a  cow  which 
had  been  killed  while  suffering  from  puerperal  fever.  The  symptoms 
were  nausea,  vomiting,  purging,  elevation  of  temperature,  and  pros- 
tration. In  some,  there  were  observed  dizziness,  sleeplessness,  and 
dilatation  of  the  pupil.  After  a  few  days  these  symptoms  gradually 
disappeared,  and  in  many  an  eczematous  eruption  of  the  lips  gave 
annoyance.  Pigs,  cats  and  dogs  that  ate  of  the  offal  of  this  animal 
were  also  made  sick.  Thorough  cooking  did  not  destroy  the  poison, 
and  those  who  took  soup  and  bouillon  made  from  the  meat  were 
affected  like  those  who  ate  of  the  muscle  fiber.  In  most  of  the  cases 
the  symptoms  came  on  within  twelve  hours  after  eating  the  meat. 

Basenau  first  found  his  bacillus  bovis  morbificans  in  the  flesh  of  a 
cow  that  was  killed  while  suffering  from  puerperal  fever,  and  later  ^ 
he  has  detected  the  same  microorganism  in  the  meat  of  animals  killed 
while  sick  with  perforative  peritonitis,  puerperal  paralysis,  and 
chronic  pyemia.  It  seems  from  the  researches  of  this  investigator 
that  there  are  varieties  of  this  germ,  the  toxins  of  some  of  which  are 
destroyed  by  heating  to  100°,  while  those  of  others  are  not.  It  is 
more  than  probable  that  Basenau's  germ  is  a  variety  of  the  colon 
bacillus  and  that  the  toxin  is  contained  within  the  cell,  and  whether 
or  not  meat  infected  with  this  organism  will  prove  harmful  depends 
upon  the  number  of  germs  present. 

In  1894,  Vaughan  and  Perkins  examined  some  dried  beef  which 
had  quite  seriously  poisoned  a  family  of  four.  There  was  nothing  in 
the  appearance  or  odor  of  the  beef  to  cause  any  suspicion.  In  fact, 
it  seemed  to  be  of  exceptionally  good  quality.  Anaerobic  cultures 
from  the  interior  of  the  meat  were  made  and  developed  a  bacillus, 
from  two  to  three  times  as  long  as  broad,  taking  the  ordinary  stains, 
motile,  with  no  spore  formation,  not  liquefying  gelatin,  but  coagulat- 
ing milk,  growing  best  at  the  temperature  of  the  body,  but  develop- 
ing its  poison  at  ordinary  temperature,  producing  gas  abundantly, 
and  pathogenic  to  white  rats,  rabbits,  and  guinea-pigs.  Sterilized 
cultures  were  also  poisonous.  Of  200  men  at  a  banquet  at  Sturgis, 
Mich.,  in  April,  1894,  everyone  who  ate  of  the  pressed  chicken 
served  was  made  ill.  Some  who  were  not  at  the  banquet  but  who 
aided  in  preparing  it,  took  small  bits  of  the  chicken  and  these  also 

^  Archiv  f.  Hygiene,  32. 


KREOTOXISMUS.  209 

were  made  sick.  All  were  taken  within  from  two  to  four  hours 
after  eating  the  chicken,  with  nausea,  violent  griping  and  purging ; 
many  fainted  while  attempting  to  rise  from  bed.  The  chickens 
were  killed  Tuesday  afternoon,  picked  and  left  hanging  in  the  mar- 
ket room  (not  in  cooling  room)  until  Wednesday  forenoon,  when 
they  were  drawn  and  carried  to  a  restaurant,  and  here  left  in  a  warm 
room  until  Thursday  morning,  when  they  were  cooked  (not  very 
thoroughly),  pressed,  and  served  at  the  banquet  that  night.  This 
food  was  examined  by  Vaughan  and  Perkins,  and  found  to  contain 
two  microorganisms,  a  slender  bacillus,  from  four  to  five  times  as 
long  as  broad,  and  a  streptococcus.  The  bacillus  was  fatal  to  white 
rats,  guinea-pigs  and  rabbits,  when  administered  intra-peritoneally, 
intra-venously,  and  subcutaneously.  The  streptococcus  was  not 
fatal  when  given  in  pure  culture,  but  mixed  cultures  of  the  two  in- 
duced death ;  and  in  these  instances,  when  administered  subcuta- 
neously, in  addition  to  lesions  found  after  the  employment  of  pure 
cultures  of  the  bacillus,  there  was  extensive  sloughing.  This  bacillus 
is  motile,  takes  the  ordinary  stains  readily,  and  is  decolorized  by 
Gram's  method.  It  grows  very  slowly  at  ordinary  temperature 
and  rapidly  at  37°.  Of  two  cultures  of  equal  age,  one  grown  at 
ordinary  temperature  and  the  other  at  37°,  1  c.c.  of  the  former  was 
necessary  to  induce  death,  while  \  c.c.  of  the  other  proved  fatal. 
The  anaerobic  cultures  were  much  more  powerful  than  the  aerobic. 
J  c.c.  of  a  beef  tea  culture  heated  to  60°  for  thirty  minutes  killed, 
while  1  c.c.  heated  to  100°  for  fifteen  minutes  failed  to  kill. 

In  an  outbreak  of  bromatotoxismus  at  an  asylum  in  Norway,  the 
patients'  food  was  veal,  and  in  this  Hoist  found  a  small  bacillus 
similar  to  but  not  identical  with  that  of  Gartner.  Lewis  found  a 
ptomain,  which  he  supposed  to  be  neuridin,  in  corned  beef  that 
poisoned  people  in  Ohio.  Poels  reported  cases  of  poisoning  in 
Rotterdam  from  the  eating  of  meat  supposed  to  be  from  a  healthy 
animal.  A  variety  of  the  colon  bacillus  was  found  in  this  meat  and 
it  was  shown  that  sterilized  cultures  were  sufficiently  toxic  to  kill 
calves.  The  same  bacillus  has  been  found  in  other  outbreaks  of 
kreotoxismus  in  Holland.  Zorkendorfer  reported  the  presence  of 
anthrax  bacilli  in  some  meat  that  poisoned  many  people,  some 
fatally,  near  Teplitz,  in  189-4  ;  however,  his  identification  of  the 
germ  cannot  be  regarded  as  positive.  Di  Mattel  has  stated  that  the 
flesh  of  animals  dead  from  symptomatic  anthrax  may  retain  its  power 
of  infection  after  having  been  preserved  in  a  dry  state  for  ten  years. 
Siedler  reported  four  cases  of  poisoning  from  decomposed  goose 
grease.  The  symptoms  consisted  of  giddiness,  prostration  and  vio- 
lent vomiting.  Christison  reported  cases  in  which  persons  were 
seriously,  some  fatally,  affected  by  eating  various  kinds  of  meat 
which  had  undergone  partial  putrefaction.  Ollivier  found  six  per- 
sons poisoned,  four  of  them  fatally,  by  eating  decomposed  mutton, 
14 


210  FOOD  POISONING. 

and  he  mentions  similar  cases  due  to  eating  ham,  Boutigmy,  hav- 
ing failed  to  find  any  poison  in  the  meat  furnished  at  a  festival,  and 
to  which  the  serious  illness  of  many  was  attributed,  made  a  meal  of 
stuffed  turkey  furnished  by  the  same  dealer,  but  after  a  short  time 
his  countenance  became  livid,  his  pulse  small  and  feeble,  a  cold 
sweat  bathed  his  body,  and  violent  vomiting  and  purging  followed. 
Geiseler  observed  nausea,  vomiting,  purging  and  delirium  after 
eating  of  bacon  which  was  imperfectly  cured.  Schroder  ^  reported 
cases  of  poisoning  which  were  due  to  the  eating  of  the  flesh  of  an  ani- 
mal killed  while  suffering  from  foot  and  mouth  disease ;  however, 
both  bacteriological  and  chemical  investigations  led  to  no  results. 
Hermann^  and  Ksensche^  found  in  some  meat  which  poisoned 
people  at  Breslau  a  typhoid-like  bacillus  whose  toxin  is  not  de- 
stroyed by  boiling.  Kuborn  *  found  a  piece  of  poisonous  meat 
which  was  infected  with  staphylococcus  pyogenes  flavus.  Johne ' 
reported  cases  of  sausage  poisoning  in  which  microorganisms  similar 
to  the  bacillus  enteritidis  were  found. 

The  most  important  contribution  that  has  recently  been  made  to 
the  subject  of  kreotoxismus  consists  of  the  discovery  by  Van  Ermen- 
gem  ^  of  a  bacillus  in  poisonous  meat  and  the  subsequent  investigation 
of  this  germ  and  its  toxin.  More  than  thirty-four  persons  were 
affected  and  three  of  these  died.  The  period  of  incubation  varied 
from  nine  to  twenty  hours,  and  the  chief  symptoms  were  pain  in  the 
stomach,  contractions  of  the  intestines,  vomiting,  obstinate  constipa- 
tion, pain  in  the  head,  fever  and  delirium,  followed  in  those  who  re- 
covered by  marked  prostration  and  muscular  weakness.  The  man, 
who  died  on  the  fifth  day,  passed  albuminous  urine,  and  section 
showed  acute  gastro-enteritis,  acute  parenchymatous  nephritis,  and 
hyperemia  of  the  lungs.  Investigation  of  the  meat  in  this  case 
showed  the  presence  of  a  bacillus  similar  to  that  found  in  other  out- 
breaks of  kreotoxismus,  but  the  greatest  interest  in  the  work  of  Van 
Ermengem  centers  about  the  cases  which  occurred  at  EUezelles,  in 
Belgium.  The  ham,  which  proved  poisonous  in  this  case,  seemed 
to  be  perfectly  normal.  Those  poisoned  showed  but  little  or  no  evi- 
dence of  gastro-intestinal  irritation,  but  nervous  symptoms  were  very 
prominent,  and  these  consisted  of  diplopia,  mydriasis,  ptosis,  aphagia, 
aphonia  and  anuria.  There  were  two  deaths.  Other  portions  of 
the  carcass  were  eaten  without  harm  and  it  was  observed  that  the 
poisonous  ham  was  the  only  part  of  the  meat  which  was  completely 
submerged  in  the  brine,  and  it  was  inferred  from  this  that  the  harm- 
ful germ  would  be  found  to  be  strictly  anaerobic.     This  inference 

J  Vierteljahrschrift  f.  gerichtl.  Medicin,  1893. 

*  Zeitschrift  f.  Fleisch-  und  Milchhygiene,  4. 
'  Zeitschriftf.  Hygiene,  22. 

*  Allgemeine.  med.  Zeitung,  1894. 

^  Bericht  iiber  das  Veterindnvesen,  1894. 

6  Ckntralblattf.  Bakteriologie,  19  ;  also  Zeitschriftf.  Hygiene,  26,  1. 


TYROTOXISMUS.  211 

proved  to  be  correct,  and  it  was  furthermore  found  that  the  bacillus 
produces  a  toxin  almost  as  poisonous  as  that  of  the  tetanus  bacillus, 
inasmuch  as  it  requires  only  0.0005  mg.  to  kill  a  rabbit  in  twenty-four 
hours.  The  microorganism  has  been  designated  as  bacillus  botu- 
linus,  and  its  poisonous  product  as  botulismustoxin.  Marinesco^ 
found  that  when  this  toxin  is  administered  by  mouth  to  cats  it 
causes  mydriasis,  partial  paralysis,  aphonia,  impossibility  of  deglu- 
tition, and  muscular  weakness.  He  found  that  the  lesions  induced 
by  this  toxin  are  confined  largely  to  the  cells  of  the  gray  matter  of 
the  cord.  With  very  small  doses  the  lesions  consist  of  a  rarefac- 
tion of  the  chromatophil  elements.  Under  the  influence  of  larger 
amounts  of  poison  the  granules  of  Nissl  undergo  more  or  less 
marked  disintegration  or  chromatolysis.  When  fatal  doses  are  ad- 
ministered some  of  these  cells  will  be  found  to  be  completely  de- 
stroyed. Brieger  and  Boer^  have  prepared  this  toxin  by  their 
method  of  precipitation  with  chlorid  of  zinc,  but  were  unable  to  ob- 
tain it  in  a  pure  state.  Apparently  it  belongs  to  the  albumoses, 
and  Kempner  ^  has  prepared  a  specific  antitoxin  by  immunizing  ani- 
mals to  the  toxin.  The  last-mentioned  observer,  together  with  Pol- 
lak,  has  confirmed  the  microscopical  studies  of  Marinesco  and  fur- 
thermore has  demonstrated  that,  under  the  influence  of  the  antitoxin, 
cells  which  have  been  injured  by  the  toxin  show  visible  evidence  of 
repair. 

Tyrotoxismus. — In  1827,  Hiinnefeld  made  analyses  of  poisonous 
cheese  and  experimented  with  extracts  upon  the  lower  animals.  He 
accepted  the  ideas  of  Kerner  in  regard  to  poisonous  sausage  in  a 
somewhat  modified  form,  and  thought  the  active  agents  to  be  sebacic 
and  caseic  acids.  About  the  same  time,  Sertiirner,  making  analyses 
of  poisonous  cheese  for  Westrumb,  also  traced  the  poisonous  principles, 
as  he  supposed,  to  these  fatty  acids.  In  1848,  Christison,  after  re- 
ferring to  the  above-mentioned  work,  made  the  following  statement : 
"  His  (Hiinnefeld' s)  experiments,  however,  are  not  quite  conclusive 
of  the  fact  that  these  fatty  acids  are  really  the  poisonous  principles, 
as  he  has  not  extended  his  experimental  researches  to  the  caseic  and 
sebacic  acids  prepared  in  the  ordinary  way.  His  views  will  probably 
be  altered  and  simplified  if  future  experiments  should  confirm  the 
late  inquiries  of  Braconnot,  who  has  stated  that  Proust's  caseic  acid 
is  a  modification  of  acetic  acid  combined  with  an  acrid  oil."  In 
1852,  Schlossberger  made  experiments  with  the  pure  fatty  acids  and 
demonstrated  their  freedom  from  poisonous  properties.  Since  the 
overthrow  of  the  fatty  acid  theory  various  conjectures  have  been 
made,  but  none  worthy  of  consideration. 

^  Comptes  rendus  de  la  Societe  de  Biologie,  1896. 

'  Deutsche  med.  Wochenschrift,  1896. 

^Deutsche  med.  Wochenschrift,  1896  ;  Zeitschrift  f.  Hygiene,  26,  481  ;  27,  213. 


212  FOOD  POISONING. 

In  the  years  1883  and  1884  there  were  reported  to  the  Michigan 
State  Board  of  Health  about  three  hundred  cases  of  cheese  poisoning. 
As  a  rule,  the  first  symptoms  appeared  within  from  two  to  four 
hours  after  eating  the  cheese.  In  a  few,  the  symptoms  were  delayed 
from  eight  to  ten  hours  and  were  very  slight.  The  attending 
physicians  reported  that  the  gravity  of  the  symptoms  varied  with 
the  amount  of  cheese  eaten,  but  no  one  who  ate  of  the  poisonous 
cheese  wholly  escaped.  One  physician  reported  the  following 
symptoms :  "  Everyone  who  ate  of  the  cheese  was  taken  with 
vomiting,  at  first  of  a  thin,  watery,  later  of  a  more  consistent 
reddish-colored  substance.  At  the  same  time  the  patient  suffered 
from  diarrhoea  with  watery  stools.  Some  complained  of  pain  in  the 
region  of  the  stomach.  At  first  the  tongue  was  white,  but  later  it 
became  red  and  dry  ;  the  pulse  was  feeble  and  irregular  ;  countenance 
pale  with  marked  cyanosis.  One  small  boy,  whose  condition  seemed 
very  critical,  was  covered  all  over  the  body  with  bluish  spots."  Not- 
withstanding the  severity  of  the  symptoms  in  many,  there  was  no 
fatal  termination  among  these  cases,  though  several  deaths  from 
cheese  poisoning  in  other  outbreaks  have  occurred.  Many  of  the 
physicians  at  first  diagnosed  the  cases  from  the  symptoms  as 
arsenical  poisoning,  and  on  this  supposition  many  of  them  adminis- 
tered ferric  hydrate.  Others  gave  alcohol  and  treated  upon  the 
expectant  plan.  Vaughan,  to  whom  the  cheese  was  sent  for 
analysis,  made  the  following  report:  "All  of  these  three  hundred 
cases  were  caused  by  the  eating  of  twelve  different  cheeses.  Of  these, 
nine  were  made  at  one  factory  and  one  each  at  three  other  factories. 
Of  each  of  the  twelve  I  received  smaller  or  larger  pieces.  Of  each 
of  ten  I  received  only  small  amounts.  Of  each  of  the  other  two 
I  received  about  eighteen  kilograms.  The  cheese  was  in  good  con- 
dition, and  there  was  nothing  in  the  taste  or  odor  to  excite  suspicion. 
However,  from  a  freshly  cut  surface  there  exuded  numerous  drops 
of  a  slightly  opalescent  fluid  which  reddened  litmus  paper  quickly 
and  intensely.  Although,  as  I  have  stated,  I  could  discern  nothing 
peculiar  in  the  odor,  if  two  samples,  one  of  good,  the  other  of 
poisonous  cheese,  were  placed  before  a  dog  or  cat,  the  animal  would 
invariably  select  the  good  cheese ;  but  if  only  poisonous  cheese  were 
offered  and  the  animal  was  hungry,  it  would  partake  freely.  A  cat 
was  kept  for  seven  days  and  fed  only  poisonous  cheese  and  water. 
It  ate  freely  of  the  cheese  and  manifested  no  untoward  symptoms. 
After  the  seven  days  the  animal  was  etherized  and  abdominal  sec- 
tion made.  Nothing  abnormal  could  be  found.  I  predicted,  how- 
ever, in  one  of  my  first  articles  on  poisonous  cheese  that  the  isolated 
poison  would  affect  lower  animals.  At  first  I  made  an  alcoholic 
extract  of  the  cheese.  After  the  alcohol  was  evaporated  in  vacuo  at 
a  low  temperature  a  residue,  consisting  mainly  of  fatty  acids,  re- 
mained.    I  ate  a  small  bit  of  this  residue,  and  found  that  it  pro- 


TYROTOXISMUS.  213 

duced  dryness  of  throat,  nausea,  vomiting  and  diarrhoea.  The  most 
of  this  extract  consisted  of  fats  and  fatty  acids  and  for  some  weeks  I 
endeavored  to  extract  the  poison  from  these  fats,  but  all  attempts 
were  unsuccessful.  I  then  made  an  aqueous  extract  of  the  cheese, 
filtered  this,  and  drinking  some  of  it,  found  that  it  also  was  poison- 
ous. But  after  evaporating  the  aqueous  extract  to  dryness  on  the 
water- bath  at  100°  the  residue  thus  obtained  was  not  poisonous. 
From  this  I  ascertained  that  the  poison  was  decomposed  or  vola- 
tilized at  or  below  the  boiling  point  of  water.  I  then  tried  distillation 
at  a  low  temperature,  but  by  this  the  poison  seemed  to  be  decomposed. 
Finally,  I  made  the  clear,  filtered  aqueous  extract,  which  was 
highly  acid,  alkaline  with  sodium  hydrate,  agitated  this  with  ether, 
removed  the  ether,  and  allowed  it  to  evaporate  spontaneously.  The 
residue  was  highly  poisonous.  By  re-solution  in  water  and  extraction 
with  ether,  the  poison  was  separated  from  foreign  substances.  As 
the  ether  took  up  some  water,  this  residue  consisted  of  an  aqueous 
solution  of  the  poison.  After  this  was  allowed  to  stand  for  some 
hours  in  vacuo  over  sulphuric  acid,  the  poison  separated  in  needle- 
shaped  crystals.  From  some  samples,  the  poison  crystallized  from 
the  first  evaporation  of  the  ether,  and  without  standing  in  vacuo. 
This  happened  only  when  the  cheese  contained  a  comparatively  large 
amount  of  the  poison.  Ordinarily,  the  microscope  was  necessary  to 
detect  the  crystalline  shape.  From  sixteen  kilograms  of  one  cheese 
I  obtained  about  0.5  gram  of  the  poison,  and  in  this  case  the  indi- 
vidual crystals  were  plainly  visible  to  the  unaided  eye.  From  the 
same  amount  of  another  cheese  I  obtained  only  about  0.1  gram,  and 
the  crystals  in  this  case  were  not  so  large.  I  have  no  idea,  how- 
ever, that  by  the  method  used  all  the  poison  was  separated  from  the 
cheese." 

To  this  substance  the  name  tyrotoxicon  (ru/?oc,  cheese,  and  ro^cxov, 
poison),  which  had  formerly  been  used  to  designate  the  undiscovered 
active  agent  in  poisonous  cheese,  was  given.  During  1887,  Wallace 
found  tyrotoxicon  in  two  samples  of  cheese  which  had  caused  serious 
illness.  The  first  of  these  came  from  Jeanesville,  Pa.,  and  the  symp- 
toms as  reported  to  Wallace  by  Doolittle,  who  had  charge  of  the 
cases,  were  as  follows  : 

Some  fifty  persons  were  affected  and  in  the  majority  of  these  the 
symptoms  appeared  within  from  two  to  four  hours,  and  consisted  of 
vertigo,  vomiting  and  severe  rigors,  varying  in  their  order  of  appear- 
ance and  severity.  Chills  and  vomiting  were  the  most  constant  and 
marked  symptoms,  and  were  soon  followed  by  pain  in  the  epigastric 
region,  cramps  in  the  feet  and  lower  limbs,  purging  and  griping  pain 
in  the  bowels,  a  sensation  of  numbness,  especially  in  the  limbs,  and 
marked  prostration,  in  some  amounting  almost  to  collapse.  The 
vomit  at  first  consisted  of  the  contents  of  the  stomach  and  had  a 
strong  cheesy  odor ;  afterward,  it  contained  mucus,  bile,  and,  in  the 


214  FOOD  POISONING. 

more  severe  cases,  blood.  The  diarrhoeal  discharges,  at  first  fecal, 
later  became  watery  and  light  colored.  No  deaths  resulted,  and  for 
the  most  part  the  effects  were  transient,  and  all  that  remained  on  the 
following  day  were  the  prostration  and  numbness,  which  disappeared 
in  from  one  to  three  days.  Children  apparently  suffered  less  than 
adults.  All  remarked  on  the  suddenness  of  the  attack,  feeling  per- 
fectly well  until  nausea  and  vertigo  set  in.  Wolff  detected  tyrotox- 
icon  in  cheese  which  poisoned  several  persons,  at  Shamokin,  Pa. 
The  pores  of  this  cheese  were  found  filled  with  a  grayish-green  fun- 
goid growth,  though  it  is  not  supposed  that  this  was  connected  in 
any  way  with  the  poisonous  nature  of  the  food.  Tests  were  made 
for  mineral  and  vegetable  poisons  with  negative  results,  after  which 
tyrotoxicon  was  recognized  both  by  chemical  and  physiological  tests. 
Ehrhart  published  the  history  of  cases  of  poisoning  from  Limburger 
cheese.  The  rind  was  covered  with  a  heavy  mould,  while  the  inte- 
rior had  become  fluid  from  putrefaction  and  was  of  bitter  taste. 
Three  ate  only  of  the  mouldy  rind,  and  these  remained  well.  The 
next  morning,  five  who  had  eaten  of  the  other  portion  suffered  from 
vertigo,  nausea,  vomiting  and  abdominal  pains;  no  stools.  The 
father  of  the  family  had  convulsive  movements  of  all  the  extremities ; 
his  pupils  were  dilated,  and  did  not  respond  to  light;  there  were 
double  vision,  cold  sweat,  cyanotic  skin,  distended  abdomen,  difficulty 
in  swallowing,  delirium,  mild  trismus,  and  a  temperature  of  40°. 
The  temperature  of  the  mother,  on  account  of  the  great  collapse,  was 
subnormal ;  she  had  no  convulsive  movements,  but  was  unconscious 
for  many  hours.  Dokkum  obtained  from  poisonous  cheese,  by  a 
modification  of  the  method  already  given  for  the  separation  of  tyro- 
toxicon, a  basic  substance,  which  when  injected  into  frogs  in  doses 
of  5  mg.  caused  paralysis,  and  death  within  thirty  minutes.  This 
investigator  thinks  that  the  base  thus  obtained  by  himself  is  not 
tyrotoxicon,  but  a  curare-like  poison  for  which  he  suggests  the  name 
tyrotoxin. 

For  some  time  after  the  discovery  of  tyrotoxicon  it  was  supposed 
that  all  cases  of  cheese  poisoning  are  due  to  this  substance,  but  sub- 
sequent investigations  have  shown  that  there  are  other  toxins  formed 
in  cheese  and  that  tyrotoxicon  is  a  somewhat  rare  poison.  In  1 890, 
Vaughan,  having  failed  to  find  any  evidence  of  tyrotoxicon  in  numer- 
ous samples  of  poisonous  cheese,  was  led  to  test  for  other  toxins. 
He  obtained  an  albumose,  forty  drops  of  an  aqueous  solution  of 
which  when  injected  under  the  skin  on  the  back  of  cats  produced 
vomiting  and  purging,  followed  by  marked  prostration  and  termi- 
nating in  some  instances  in  death.  This  substance  belongs  to  the  so- 
called  poisonous  albumins.  From  its  aqueous  solution  it  is  not  precip- 
itated by  heat  or  nitric  acid,  singly  or  combined.  It  is  not  precipi- 
tated by  saturation  with  sodium  sulphate,  nor  by  a  current  of  carbonic 
acid  gas  ;  therefore  it  is  not  a  globulin.     It  is  precipitated  by  satura- 


OALACTOTOXISMUS.  215 

tion  with  ammonium  sulphate,  and  this  fact  distinguishes  it  from  the 
peptons.  In  1895  Vaughan  and  Perkins  obtained  from  a  piece  of 
cheese  which  had  proved  fatal  to  one  man,  two  bacilli,  one  of  which 
elaborates  an  active  toxin  ;  both  filtered  and  heated  cultures  kill  ani- 
mals promptly.  Yaughan  and  McClymonds  ^  examined  sixty-five 
samples  of  cheese  from  as  many  different  manufacturers.  Of  these, 
forty-nine  were  what  is  ordinarily  known  as  American  green  cheese. 
These  were  made  in  Michigan,  Wisconsin,  Illinois,  New  York, 
and  Canada.  Eight  of  the  forty-nine  samples  were  sent  to  the 
laboratory  because  persons  eating  them  had  suffered  from  nausea, 
vomiting  and  purging ;  the  other  samples  were  not  known  to  be 
poisonous.  Everyone  of  the  forty-nine  samples  of  American  green 
cheese  furnished  cultures  which  killed  white  rats,  guinea-pigs 
and  rabbits.  The  toxicogenic  germ  in  all  these  samples  belongs  to 
the  colon  group,  and  whether  or  not  a  given  sample  of  green  cheese 
unpleasantly  aifects  the  consumer  will  depend  upon  the  amount  and 
virulence  of  the  germ  present  in  the  cheese.  Yaughan  and  Cooley  ^ 
have  shown  that  the  colon  toxin  is  contained  in  the  germ  cell  from 
which  it  does  not,  at  least  under  ordinary  conditions,  diffuse  into  the 
culture  medium.  This  toxin  may  be  heated  in  water  to  a  very  high 
temperature  without  destruction  of  its  toxicity,  as  is  illustrated  by 
the  following  experiment :  200  mg.  of  the  crude  toxin  was  placed 
with  10  c.c.  of  water  in  a  tube  which  was  sealed  and  heated  to  184° 
for  thirty  minutes.  On  opening  the  tube  the  content  was  found  to 
be  milky  and  microscopical  examination  showed  a  granular  mass 
containing  a  few  unbroken  cells.  Portions  of  this  heated  substance 
injected  into  guinea-pigs  caused  death,  and  autopsy  showed  the 
lesion  usually  found  in  these  animals  when  killed  with  colon  toxin. 
Apparatus  has  been  devised  for  obtaining  the  colon  toxin  in  large 
amount  ^  and  it  has  been  found  that  with  a  very  virulent  culture  the 
powdered  germ  is  often  sufficiently  toxic  to  kill  guinea-pigs  of  200 
gram  weight  in  doses  of  ^  mg. 

Galactotoxismus. — Tyrotoxicon  has  been  found  in  milk  in  numer- 
ous instances,  having  first  been  detected  in  this  fluid  in  1885,  soon 
after  its  discovery  in  cheese.  In  1886  Newton  and  "Wallace  detected 
this  poison  in  milk  which  seriously  affected  a  large  number  of  persons 
at  Long  Branch.  The  poisonous  milk  came  solely  from  one  dairy- 
man and  investigation  showed  the  following  condition  of  affairs  : 
The  cows  were  milked  at  the  unusual  hours  of  midnight  and  noon,  and 
the  noon's  milk — that  which  alone  was  followed  by  illness — ^was 
placed  while  hot  in  the  cans,  and  then,  without  any  attempt  at  cool- 
ing, carted  eight  miles  during  the  warmest  part  of  the  day  in  a  very 
hot  month.     During  this  time  the  unknown  germ  which  elaborates 

'  Jacobi  Festschrift,  p.  108. 

^  Journal  of  the  American  Medical  Association,  1901,  also  Amencan  Medicine,  1901. 

^  Transactions  of  the  Association  of  American  Physicians,  1901. 


216  FOOD  POISONING. 

tyrotoxicon  undoubtedly  grew  abundantly  in  the  milk  and  its  toxin 
was  easily  detected  by  both  chemical  and  physiological  tests.  In  the 
same  year  Schearer  found  tyrotoxicon  in  milk  used  by,  and  in  the 
vomited  matter  of,  persons  made  sick  at  a  hotel  at  Corning,  la.  In 
1887,  Firth,  an  English  army  surgeon,  stationed  in  India,  reported 
an  outbreak  of  milk  poisoning  among  the  soldiers  of  his  garrison. 
From  the  milk  he  separated,  by  the  method  already  given,  tyrotoxi- 
con and  demonstrated  its  action  upon  the  lower  animals.  In  the 
same  year  Vaughan  reported  the  Milan  cases  of  milk  poisoning, 
three  of  which  terminated  fatally.  His  report  of  his  observation  of 
the  symptoms  is  as  follows :  I  first  saw  these  patients,  Sunday,  Sep- 
tember 25th;  on  a  sofa  in  the  room  we  found  the  daughter,  who  had 
been  vomiting  during  the  day  and  seemed  much  exhausted.  She 
was  not  inclined  to  talk,  and  seemed  to  be  in  a  stupor,  though  when 
spoken  to  she  responded  rationally.  Her  pupils  were  slightly  dilated, 
her  tongue  coated,  her  pulse  120  and  weak;  her  face  pale;  and  a 
violent  throbbing  could  be  felt  over  the  abdomen,  which  was  re- 
tracted. Her  temperature  was  96°  F.  In  another  room  were  the 
father,  mother  and  son,  two  of  them  dying.  The  father  was  rational 
and  talked  with  some  freedom,  when  asked  concerning  the  kind  of 
food  they  had  been  eating.  His  pupils  were  normal,  his  face  could 
not  be  said  to  present  any  peculiar  feature,  his  pulse  was  rapid, 
breathing  somewhat  rapid,  and  the  throbbing  in  the  abdominal  area 
was  plainly  felt.  The  abdomen  was  retracted  and  there  was  no 
pain  on  pressure.  He  complained  of  a  burning  constriction  of  the 
throat,  swallowed  with  difficulty,  and  said  that  his  throat  and  stom- 
ach felt  as  though  they  were  on  fire.  The  mother  lay  with  eyelids 
closed  as  if  in  a  deep  sleep.  Her  pulse  was  rapid,  her  face  had  a 
livid  flush,  her  breathing  was  about  35  per  minute,  and  labored. 
The  skin  was  cool,  but  neither  abnormally  moist  nor  specially  dry 
and  harsh.  She  could  not  be  aroused.  In  fact,  she  was  comatose. 
The  son  rolled  uneasily  from  one  side  of  the  bed  to  the  other.  His 
breathing  also  was  labored.  His  eyelids  were  closed,  and  the  pupils 
were  markedly  dilated — did  not  respond  to  light.  He  could  not  be 
aroused.  In  mother  and  son,  as  well  as  in  father  and  daughter,  the 
abdomen  was  retracted,  and  the  throbbing  in  the  abdominal  area  was 
easily  felt. 

The  symptoms  were  not  those  of  morphin,  strychnin,  digitalis,  or 
aconite.  They  did  have  some  resemblance  to  those  of  belladonna 
but  were  not  identical ;  the  pupils  were  not  so  widely  dilated  as  they 
are  in  belladonna  poisoning  ;  there  was  in  none  of  these  persons  the 
active  delirium  of  belladonna  poisoning  ;  there  was  no  picking  at  the 
clothing  ;  no  grasping  of  imaginary  objects  in  the  air ;  no  hallucina- 
tions of  vision. 

The  family,  which  consisted  of  the  four  sick  persons  and  of  a 
daughter  about  twenty  years  of  age,  who  was  away  from  home  at  the 


GALACTOTOXISMUS.  217 

time  when  the  others  were  taken  ill  and  for  some  months  before  that 
time,  was  evidently  a  tidy  one.  This  was  shown  by  their  personal 
appearance  and  by  the  clothing  and  bedding ;  but  the  house  in  which 
they  lived  was  old,  and  much  decayed.  One  corner  of  one  of  the 
rooms  had  been  transformed  into  a  buttery,  and  in  this  the  food  was 
kept  on  shelves.  On  account  of  the  more  frequent  scouring  de- 
manded by  that  part  of  the  house  the  boards  constituting  the  floor 
had  rotted  away,  and  a  second  layer  had  been  placed  over  the  original 
floor.  Between  these  two  floors  there  was  found  a  great  mass  of 
moist,  decomposing  matter,  the  accumulation  of  years,  which  the 
broom  could  not  reach.  When  this  floor  was  taken  up,  a  peculiar, 
nauseating  odor  was  observable,  and  was  sufficient  to  cause  actual 
vomiting  in  one  of  the  persons  engaged  in  the  examination.  Tyro- 
toxicon  was  found  in  milk  which  had  been  used  by  the  family  and 
had  stood  in  this  room,  and  it  was  also  produced  in  sterilized  milk 
by  inoculating  it  with  bits  of  earth  taken  from  between  the  floors. 
An  autopsy  held  on  one  of  the  fatal  cases  showed  as  the  most  marked 
abnormality  tightly  constricted  areas  of  the  large  intestine  such  as  is 
sometimes  seen  in  lead  poisoning,  and  which  had  been  quite  gener- 
ally observed  in  the  lower  animals  experimentally  killed  by  the  ad- 
ministration of  tyrotoxicon.  Novy  tested  a  cold-water  extract  of 
the  finely-divided  intestines  for  ptomains.  The  fluid,  which  was 
acid  in  reaction  was  filtered,  then  neutralized  with  sodium  carbon- 
ate, and  shaken  with  ether.  The  ether,  after  separation,  was  re- 
moved and  allowed  to  evaporate  spontaneously.  The  residue  was 
dissolved  in  water  and  extracted  again  with  ether.  This  ether  resi- 
due gave  the  chemical  reactions  for  tyrotoxicon  and  a  portion  of  it 
was  administered  to  a  kitten  about  two  months  old.  Within  one- 
half  hour  the  kitten  began  to  retch  and  soon  it  vomited,  and  within 
the  next  three  hours  it  vomited  as  many  as  five  times.  There  was 
no  purging  but  the  retching  and  heavy  breathing,  with  evidences  of 
prostration,  continued  more  or  less  marked  for  two  days,  after  which 
the  animal  slowly  recovered.  A  quantity  of  fresh  milk  was  divided 
into  five  portions  of  one  quart  each,  placed  in  bottles  which  had 
been  thoroughly  cleansed,  and  treated  in  the  following  manner  :  No. 
1  consisted  of  the  milk  only,  and  was  employed  as  a  control  test. 
No.  2  was  mixed  with  a  drachm  of  vomited  matter.  No.  3  was 
treated  with  a  portion  of  the  contents  of  the  stomach.  No.  4  was 
treated  with  an  aqueous  extract  of  the  intestine.  No.  5  was  treated 
with  a  small  portion  of  the  soil  which  had  been  taken  from  the  floor 
of  the  buttery,  stirred  up  with  water. 

These  bottles  were  placed  in  an  air-bath  and  kept  at  a  tempera- 
ture of  from  25°  to  30°  for  twenty-four  hours,  and  then  each  was 
tested  for  tyrotoxicon,  the  result  in  No.  1  being  negative,  while  in 
all  of  the  others  it  was  positive.  These  tests  were  both  chemical 
and  physiological.     All  the  samples  yielded  a  non-poisonous  base 


218  FOOD  POISONING. 

when  treated  according  to  Brieger's  method,  and  the  same  substance 
was  obtained  from  perfectly  fresh  milk.  It  was  most  probably- 
formed  by  the  action  of  the  heat  and  reagents  employed  in  this 
method.  This  base  was  obtained  in  crystalline  form,  and  several 
portions  of  it  were  administered  to  kittens  without  effect.  Tyro- 
toxicon  was  obtained  from  the  filtered  milk  by  two  methods  :  (1) 
The  method  which  has  been  previously  described,  and  which  con- 
sists in  neutralizing  the  filtered  milk  with  sodium  carbonate  and  ex- 
tracting with  ether.  That  portion  of  the  poison  employed  in  the 
physiological  tests  was  obtained  in  this  way,  and  in  order  to  be  sure 
that  no  poison  came  from  the  ether,  the  extract  from  the  milk  to 
which  nothing  had  been  added,  was  given  to  a  kitten,  and  was 
found  to  produce  no  effect.  (2)  The  filtrate  from  the  milk  was 
heated  to  70°  for  some  minutes  and  refiltered.  This  filtrate,  which 
was  perfectly  clear,  was  treated  with  a  small  quantity  of  nitric  acid 
in  order  to  convert  the  tyrotoxicon  into  a  nitrate.  Then  pure 
potassium  hydrate  in  the  solid  form  was  added  until  the  solution 
was  strongly  alkaline.  This  solution  was  concentrated  so  far  as  it 
could  be  on  the  water-bath.  (The  potassium  compound  of  tyrotoxi- 
con is  not  decomposed  below  130°.)  The  dark  brown  residue,  after 
cooling,  was  examined  with  the  microscope  and  found  to  contain 
the  crystalline  plates  of  tyrotoxicon  potassium  hydrate,  along  with 
the  prisms  of  potassium  nitrate.  The  former  was  separated  from 
the  latter  by  extraction  with  absolute  alcohol  and  filtration.  The 
alcohol  was  evaporated  to  dryness  on  the  water-bath,  and  the  res- 
idue again  extracted  with  alcohol.  From  this  alcoholic  solution 
tyrotoxicon  was  precipitated  with  ether.  The  precipitate  was  decom- 
posed by  adding  acetic  acid  and  heating,  the  tyrotoxicon  being 
broken  up  into  nitrogen  and  phenol.  The  phenol  was  recognized 
by  precipitation  with  bromine  water,  and  by  other  well-known  tests. 

The  coroner's  jury,  after  hearing  the  testimony  briefly  outlined 
above,  rendered  a  verdict  of  death  from  poisoning  with  tyrotoxicon, 
and,  so  far  as  we  know,  this  is  the  first  instance  in  which  a  jury  has 
rendered  a  verdict  of  death  due  to  a  putrefactive  poison. 

Camman  reported  twenty-three  cases  of  milk  poisoning  which  he 
attributed  to  tyrotoxicon,  although  this  substance  could  not  be  found 
in  the  milk,  and  Kinnicutt  has  isolated  tyrotoxicon  from  milk  which 
had  been  kept  for  some  hours  in  unclean  vessels. 

Gaffky  has  reported  a  case  of  enteritis,  closely  simulating  typhoid 
fever,  which  was  due  to  the  presence  of  a  virulent  form  of  the  colon 
bacillus  in  the  milk  consumed  by  the  individual.  The  cow  that 
gave  this  milk  was  at  the  time  suffering  from  a  bloody  diarrhoea, 
and  it  is  more  than  probable  that  some  of  the  liquid  discharged 
from  the  animal  fell  upon  the  udder  or  in  some  other  manner  found 
its  way  into  the  milk.  Rehn  has  reported  similar  cases  and  has  de- 
tected the  colon  bacillus  in  the  milk  taken  by  the  sick. 


GALACTOTOXISMUS.  219 

Ice  cream,  frozen  custard,  cream  puffs,  and  other  articles  of  food 
consisting  largely  of  milk,  are  frequently  harmful.  The  injurious 
effects  of  these  substances  have  been  attributed  to  plants  eaten  by 
the  animals,  and  the  flavoring  and  coloring  matters  used  in  the  prep- 
aration of  these  foods.  Even  within  recent  years  the  claim  has 
been  put  forward  that  ice  cream  poisoning  is  due  to  artificially  pre- 
pared vanillin,  but  vanilla  extracts  used  in  the  preparation  of  foods, 
which  prove  to  be  poisonous,  have  been  swallowed  in  large  quanti- 
ties by  chemists  and  have  been  administered  to  animals  without  the 
slightest  effect.  It  has  also  been  claimed  that  ice  cream  often  owes 
its  poisonous  properties  to  small  quantities  of  zinc  or  tin  dissolved 
during  the  process  of  freezing.  This  statement  is  perfectly  absurd 
when  we  find,  as  we  frequently  do,  that  a  sample  of  ice  cream  will 
act  more  powerfully  as  an  emetic  than  will  sulphate  of  zinc,  grain  for 
grain.  It  is  within  the  range  of  possibility  that  poisonous  extracts 
may  be  used  in  flavoring  milk  preparations  and  it  is  a  well-known 
fact  that  chromate  of  lead  has  been  found  in  cream  puffs.  But  it  is 
certainly  true  that  neither  flavoring  agent  nor  metals  are  account- 
able for  the  inj  urious  effects  observed  to  follow  the  eating  of  poison- 
ous ice  cream  and  similar  milk  products.  Moreover,  ice  cream 
flavored  with  chocolate  and  that  flavored  with  lemon  have  also  been 
observed  to  be  poisonous,  and  vanilla  ice  cream  is  more  frequently 
poisonous  for  the  very  good  reason  that  this  flavoring  is  used  more 
largely  than  any  other,  and  possibly  than  all  others  combined. 

Vaughan  and  Novy  have  found  tyrotoxicon  in  numerous  samples 
of  poisonous  ice  cream  and  custard.  Schearer  reported  the  same 
poison  in  both  vanilla  and  lemon  ice  cream  which  made  many  sick 
at  Nugent,  la.  Allaben  observed  numerous  cases  poisoned  with 
lemon  ice  cream,  and  Welford  has  obtained  tyrotoxicon  from  custard 
flavored  with  lemon.  It  must  not  be  inferred,  however,  that  this  is 
the  only  toxin  that  is  found  in  ice  cream  and  other  milk  compounds. 
In  1896,  Vaughan  and  Perkins^  reported  the  detection  of  a  new 
toxin  in  both  ice  cream  and  cheese.  This  substance  differs  from 
tyrotoxicon  chemically  inasmuch  as  it  is  not  removed  from  alkaline 
solutions  by  extraction  with  ether.  Physiologically  its  action  on  the 
heart  closely  resembles  that  of  muscarin  or  neurin.  Pathologically 
it  induces  a  high  degree  of  local  inflammation  when  injected  subcu- 
taneously  or  intra- peritoneally ;  and  after  death  the  contractions  of 
the  intestines  so  characteristic  of  tyrotoxicon  poisoning  were  never 
found,  although  more  than  200  animals  were  experimented  upon  in 
this  investigation.  In  the  persons  poisoned  with  this  food,  s\Tnptoms 
appeared  within  from  three  to  six  hours  and  at  first  consisted  of 
nausea  which  in  all  instances  was  followed  by  vomiting.  Diarrhoea 
was  present  in  the  majority  but  not  in  all.  The  vomiting  was  ac- 
companied by  sharp  pains  through  the  abdomen,  and  it  is  stated  that 

^Archivf.  Hygiene,  27. 


220  FOOD  POISONING. 

in  some  the  pain  was  partially  relieved  by  strong  pressure.  The 
most  alarming  phenomenon  to  the  physicians  in  attendance  was 
feebleness  of  the  heart's  action.  The  hands  and  feet  grew  cold,  then 
the  entire  body  became  cool  and  clammy,  and  in  many  the  radial 
pulse  was  not  perceptible.  This  condition,  together  with  a  heavy 
stupor  in  some,  gave  occasion  for  alarm,  and  hypodermic  injections 
of  brandy,  digitalis,  strychnin  and  nitroglycerin  were  employed,  each 
physician  selecting  the  stimulant  in  which  he  had  the  most  confidence, 
or  taking  that  which  he  had  at  hand.  In  one  instance  the  patient 
became  wildly  delirious,  crying  out  and  attempting  to  rise  from  the 
bed.  Those  who  vomited  but  little  and  had  no  diarrhoea  fell  into  a 
heavy  stupor,  and  it  is  highly  probable  that  these  were  in  greater 
jeopardy  than  the  others.  The  early  and  thorough  vomiting  doubt- 
less was  the  most  potent  agent  in  saving  those  who  had  taken  the 
larger  quantities  of  the  infected  food.  The  toxin  formed  by  the  germ 
found  in  this  food  is  not  destroyed  by  boiling.  The  germ  which 
produced  this  toxin  bears  a  close  resemblance  to  the  colon  bacillus, 
but  differs  from  a  typical  member  of  this  group  in  the  following 
particulars :  (1)  The  new  bacillus  failed  to  give  the  indol  reaction. 
(2)  Both  coagulate  milk,  but  the  new  germ  acts  more  promptly  than 
the  colon  bacillus.  (3)  The  pleasant  butyric  ether  odor  of  milk  cul- 
tures of  the  new  bacillus  is  not  developed  in  cultures  of  the  colon 
bacillus  in  the  same  medium.  (4)  The  new  germ  grows  abundantly 
on  carrots  forming  a  creamy  layer,  and  gives  ofP  a  sour  odor ;  while 
the  colon  bacillus  grows  much  less  vigorously  and  gives  off  no  sim- 
ilar odor. 

Undoubtedly  there  are  many  forms  of  the  colon  bacillus  which 
frequently  find  their  way  into  milk  and,  on  account  of  the  toxin  con- 
tained within  their  cells,  they  render  this  and  various  other  foods 
of  which  milk  is  a  constituent  more  or  less  poisonous. 

Sitotoxismus. — Under  the  heading  of  sitotoxismus  we  may  include 
all  forms  of  poisoning  with  vegetable  foods  infected  with  moulds  and 
bacteria.  All  sitotoxicons  are  not  bacterial  products  ;  however,  for 
completeness  we  will  briefly  review  the  entire  subject,  excluding,  of 
course,  all  cases  of  poisoning  due  to  admixture  with  mineral  sub- 
stances. We  shall  also  attempt  to  exclude  as  far  as  possible  all  dis- 
cussion of  plants  that  are  in  and  of  themselves  poisonous. 

Ergotismus,  sometimes  called  ergotism,  is  due  to  poisoning  with  a 
fungus  known  as  claviceps  purpurea,  which  develops  in  the  flowers 
of  rye,  other  grains,  and  certain  wild  grasses.  It  is  most  frequently 
found  in  rye  and  darnel.  Early  in  the  development  of  the  rye  flower 
there  may  appear  in  its  interior  a  sweet,  unpleasant-smelling  liquid 
which  sometimes  forms  so  abundantly  that  it  overflows,  runs  down 
upon  the  stalk,  and  falls  upon  the  ground.  The  sugar  which  it  con- 
tains attracts  ants  and  other  insects,  and  these  aid  in  the  distribution 


SITOTOXISM  US.  22 1 

of  the  fungus.  There  are  certain  conditions  which  are  known  to 
favor  the  development  of  this  parasite.  It  is  more  common  when 
there  is  a  rainy  spring  followed  by  a  hot,  dry  summer.  Thorough 
cultivation  of  the  soil  kills  the  parasite,  and  for  this  reason  ergot  is 
more  abundant  in  countries  where  the  soil  is  not  well  cultivated,  and 
ergotism  has  within  recent  years  prevailed  in  epidemic  form  only  in 
Russia  and  in  Spain.  Grains  of  ergot,  after  having  been  exposed  to 
the  air  for  a  few  months,  lose  in  large  part  their  toxicity,  and  con- 
sequently epidemics  of  ergotism  follow  closely  upon  the  harvests, 
and  especially  upon  poor  harvests,  when  the  parasite  is  most  abund- 
ant and  the  people  are  compelled  to  feed  upon  what  they  have  with- 
out close  inquiry  as  to  its  quality.  However,  it  may  be  pointed  out 
here  that  in  the  present  state  of  civilization  there  is  but  little  excuse 
for  the  existence  of  epidemics  of  ergotism.  In  the  first  place,  thorough 
cultivation  of  the  soil  would  soon  completely  eradicate  this  mould, 
and  a  proper  selection  of  seed  would  do  much  in  the  same  direction. 
As  early  as  1858  Kiihn  pointed  out  the  benefit  that  would  be  secured 
by  an  early  harvesting  of  fields  contaminated  with  ergot,  as  by  this 
means  the  spread  and  consequent  development  of  this  parasite  would 
be  largely  prevented.  Moreover,  the  ergot  grain  is  much  larger 
than  that  of  rye,  and  this  difference  in  size  permits  of  the  easy  sep- 
aration of  the  two  by  means  of  sieves  especially  constructed  for  this 
purpose.  The  commercial  value  of  ergot  is  so  much  greater  than 
that  of  rye  that  the  time  given  to  the  separation  of  the  two  would  be 
profitably  spent,  and  yet  so  dense  is  the  ignorance  and  so  pronounced 
is  the  indolence  of  certain  peasant  classes  that  epidemics  of  ergotism 
continue  and  probably  will  continue  for  many  years. 

Kobert  and  his  student,  Griinefeld,  have  found  three  poisons  in 
ergot;  these  are  ergotinic  acid,  sphacelinic  acid  and  cornutin.  The 
first  of  these,  ergotinic  acid,  is  poisonous  when  injected  subcutane- 
ously  or  intravenously,  but  seems  to  be  devoid  of  harmful  properties 
when  taken  by  the  mouth,  hence  it  can  play  no  part  in  the  causation 
of  ergotism.  In  all  cases  of  ergotism  both  the  sphacelinic  acid  and 
the  cornutin  are  contained  in  the  ergot ;  therefore,  a  clinical  picture 
of  the  disease  must  be  a  composite  resulting  from  the  combined 
action  of  the  two,  and  it  must  vary  with  the  preponderance  of  one  or 
the  other  in  the  ergot  taken. 

It  is  believed  that  sphacelinic  acid  is  the  constituent  of  ergot  that 
causes  gangrene  and  develops  the  cachexia  of  the  disease.  Griine- 
feld fed  animals  with  sphacelinic  acid  and  induced  gangrene  in  all ; 
in  cocks,  the  comb,  then  the  wattles,  tongue,  wings  and  crop,  re- 
spectively were  affected ;  in  hogs,  the  ears  fell  oflf,  bit  by  bit ;  in 
horses  and  cows,  the  tails,  ears  and  hoofs  separated ;  while  in  dogs 
and  cats  the  gangrene  usually  began  in  the  skin.  When  locally  ap- 
plied in  concentrated  solution  sphacelinic  acid  causes  gangrene  of 
the  tissues  with  which  it  comes  in  contact  and    this  explains  the 


222  FOOD  POISONING. 

necrosis  of  the  living  tissue,  the  ulcerations,  and  the  hemorrhages 
into  the  intestines. 

Cornutin  does  not  cause  death  of  tissue,  but  acts  directly  upon  the 
nervous  system,  and  is  believed  to  be  the  active  agent  in  the  causa- 
tion of  ergotismus  convulsivus.  It  acts  on  the  brain  and  cord, 
affecting  the  vagus  and  vasomotor  centers,  and  acting  through  the 
lumbar  cord  upon  the  uterus.  Cornutin  readily  undergoes  decom- 
position, gradually  losing  its  virulence,  and  is  found  only  in  fresh 
ergot,  disappearing  more  quickly  than  sphacelinic  acid.  For  this 
reason  it  happens  that  those  symptoms  due  to  cornutin  are  more 
prominent  in  outbreaks  occurring  soon  after  the  harvest ;  while  those 
due  to  sphacelinic  acid  are  seen  in  both  early  and  late  epidemics. 

There  are  some  reasons  for  believing  that  there  are  bacterial  prod- 
ucts formed  in  ergotized  bread,  and  to  these  has  been  attributed  the 
septic  character  of  certain  epidemics  of  ergotism.  However,  this  is  a 
mere  supposition  and  there  has  been  no  scientific  experimentation 
made  in  its  support.  It  is  easy  to  see  how  sepsis  occurs  in  ergotism 
without  the  necessity  of  supposing  the  presence  of  bacterial  products 
in  ergotized  bread.  In  gangrene  of  the  intestines  bacterial  infection 
through  the  diseased  intestinal  walls  may  easily  occur ;  so  in  gan- 
grene of  the  skin  infection  from  without  may  take  place  with  equal 
readiness. 

Lathyrismus,  or  lathyrism,  is  a  form  of  spastic  spinal  paralysis  due 
to  intoxication  from  the  eating  of  the  seeds  of  certain  species  of  the 
genus  Lathyrus  of  the  vetch  tribe.  Of  the  more  than  120  known 
species  of  lathyrus,  13  are  native  to  the  United  States,  and  others 
are  cultivated  here  on  account  of  their  showy  flowers,  the  sweet  pea 
of  the  garden  being  an  example  of  the  latter.  In  northern  Africa 
and  southern  Europe  lathyrism  has  been  frequently  observed,  and  it 
occasionally  occurs  in  India  aud  other  parts  of  Asia.  The  literature 
of  lathyrism  shows  that  this  disease  was  formerly  much  more  prev- 
alent than  it  is  at  present.  As  early  as  1671  it  was  known  that 
bread  made  of  vetch  seeds  mixed  with  graham  seriously  affected 
those  who  ate  of  it  for  any  length  of  time  and  the  Grand  Duke  of 
Wiirtemberg  issued  an  edict  forbidding  the  use  of  food  of  this  kind. 
It  was  then  noticed  that  those  who  ate  of  this  bread  suffered  from 
marked  stiffness  of  the  extremities  and  the  disease  was  regarded  as 
incurable,  although  death  seldom  resulted  from  it.  Numerous  at- 
tempts have  been  made  to  isolate  the  poisonous  principle  or  prin- 
ciples of  lathyrus,  but,  so  far,  the  results  obtained  have  been  unsat- 
isfactory and  to  some  extent  contradictory.  Teilleux  obtained  a 
resinous  body  which,  when  administered  to  rabbits  in  gram  doses, 
caused  tetanic  movements  of  the  muscles  and  finally  paralysis  of  the 
posterior  extremities,  death  occurring  within  four  days.  From 
lathyrus  cicera,  Bourlier  obtained  an  extract  which  killed  frogs  and 


MA'iDISMUS.  223 

small  birds  within  forty-eight  hours  at  most.  An  alkaloidal  body- 
was  obtained  by  Marie  from  the  seeds  of  the  lathyrus  sativa ;  how- 
ever, this  substance  when  administered  subcutaneously  to  guinea- 
pigs  does  not  induce  any  of  the  characteristic  symptoms  of  lathy rism. 
Astier  obtained  an  alcoholic  extract  which,  after  repeated  injections, 
induced  in  dogs  complete  paraplegia,  from  which  the  animal  slowly 
recovered.  There  are  good  reasons  for  believing  that  whatever 
the  poisonous  substance  may  be,  it  is  destroyed  at  a  high  tempera- 
ture. The  Arabs  of  northern  Africa  eat  vetch  prepared  in  two 
ways:  One  preparation,  known  as  '^Kouskouson,"  is  steamed  or 
boiled,  while  the  other  dish,  known  as  "  Galette,"  is  cooked  at  a 
higher  temperature,  and  it  is  said  to  be  a  well  authenticated  fact  that 
injurious  effects  more  frequently  follow  the  use  of  the  former  than  of 
the  latter.  In  man  the  first  symptom  of  lathyrism  usually  mani- 
fested is  a  chill,  which  is  followed  by  pain  in  the  loins  and  legs.  A 
girdle  sensation  is  complained  of  by  some,  and  motor  lameness  of 
the  lower  extremities  is  common.  The  patient  walks  with  difficulty, 
and  later  finds  locomotion  wholly  impossible.  The  knee  reflex  is 
greatly  intensified,  and  a  paresthesia  with  formication  may  be  marked. 
It  is  claimed  by  some  that  gangrene  of  the  feet  and  legs  may  occur, 
but  it  is  possible  that  cases  upon  which  this  statement  is  founded 
were  due  to  ergot  poisoning.  The  old  belief  that  recovery  never 
occurs  is  not  supported  by  more  recent  observation,  and  many  of  the 
milder  cases  are  greatly  improved  by  proper  medicinal  treatment. 

Maidismus. — Ordinarily  known  as  pellagra,  this  may  be  defined 
as  a  progressive  disease  leading  to  paralytic  and  other  nervous  dis- 
orders, and  caused  by  intoxication  from  the  eating  of  damaged 
Indian  corn.  The  geographical  distribution  of  maidismus  is  con- 
fined to  that  portion  of  Europe  lying  between  the  parallels  of  42° 
and  48°  N.,  with  the  exception  of  Corfu,  one  of  the  Ionian  islands, 
but  within  the  above-mentioned  region  this  disease  is  by  no 
means  uniformly  or  universally  distributed.  It  prevails  in  some 
localities  to  such  an  extent  that  it  has  become  a  national  calamity. 
In  1879,  one  hundred  thousand  cases  of  this  disease  were  reported 
in  Italy,  and  in  1881,  fifty-six  thousand  in  Lombardy  alone. 
Pellagra  is  confined  to  countries  where  the  staple  article  of  diet  is 
maize,  and  yet  Indian  corn  constitutes  a  most  nutritious  and  health- 
ful article  of  food  in  other  countries,  as  has  been  abundantly  demon- 
strated by  the  former  well  nourished  condition  of  the  large  colored 
population  of  the  southern  United  States,  for  there  probably  never 
has  been  a  class  of  day  laborers,  certainly  never  a  class  of  servants, 
better  fed  and  nourished  than  were  the  negroes  of  the  South  before 
their  emancipation ;  and  corn  bread,  made  from  mature  corn  and 
properly  prepared,  is  both  healthy  and  nutritious.  Pellagra  is 
known  only  in  those  countries  where,  on  account  of  an  uncongenial 


224  FOOD  POISONING. 

climate,  or  from  barrenness  of  the  soil,  or  from  lack  of  proper 
cultivation,  maize  does  not  mature. 

While  there  can  no  longer  be  any  doubt  that  pellagra  is  an  intoxi- 
cation due  to  poison  formed  in  corn  meal  or  bread,  we  have,  as  yet, 
no  positive  information  concerning  either  the  ferment  which  causes 
these  harmful  changes  or  the  poisonous  substance  or  substances  that 
are  formed.  Some  think  that  the  disease  is  an  intestinal  mycosis, 
due  to  infection  with  a  parasitic  mould,  which  is  introduced  into  the 
body  with  this  food.  Carboni  found  in  the  damaged  meal  used  by 
pellagrous  persons,  also  in  their  feces,  a  bacterium  to  which  he  has 
given  the  name  bacillus  maidis,  and  to  which  he  ascribes  the  dis- 
ease. Majocchi  claims  to  have  found  this  germ  in  the  blood  of 
pellagrous  individuals,  and  according  to  Paltauf  and  Heider  the 
grains  of  corn  become  infected  during  the  wet  season  with  the 
bacillus  maidis  and  the  bacillus  mesentericus  fuscus,  and  these  de- 
compose the  moist  meal  producing  ptomains  which  constitute  the 
toxins.  Others  claim  that  the  so-called  bacillus  maidis  is  nothing 
more  than  the  widely  distributed  potato  bacillus,  that  it  is  incapable 
of  generating  toxins  under  any  conditions  and  that  it  is  by  no 
means  constantly  found  in  the  intestines  of  pellagrous  individuals. 
Lombroso  thinks  the  disease  is  an  intoxication  rather  than  an  infec- 
tion, and  believes  that  it  is  due  to  certain  chemical  poisons  formed 
by  bacterial  activity.  This  investigator  has  obtained  from  powdered 
corn  which  has  been  allowed  to  ferment  at  from  25°  to  30°  for 
twenty-four  to  thirty-six  hours,  an  alcoholic  extract  and  an  oily 
substance,  and  with  these  he  thinks  that  he  has  induced  the 
characteristic  symptoms  of  pellagra  in  man  and  animals.  The 
alcoholic  extract  of  this  corn  contains  a  basic  substance  or  sub- 
stances to  which  Lombroso  has  applied  the  name  pellagrocein,  and 
according  to  his  theory  there  are  two  toxins,  the  combined  action  of 
which  gives  rise  to  the  complex  symptoms  of  pellagra,  similar  to 
the  action  of  sphacelinic  acid  and  cornutin  in  ergotism.  One  of 
these  poisons  he  thinks  has  a  strychnin-like  effect,  while  the  other  is 
narcotic  in  its  action.  Neusser  believes  that  there  is  nothing 
directly  harmful  in  the  food  when  it  is  taken  into  the  body,  but 
that  poisons  are  formed  in  the  intestines  and  he  makes  the  disease  a 
specific  form  of  auto-intoxication.  It  is  claimed  that  sporadic  cases 
of  pellagra  may  be  due  to  the  use  of  whisky  made  from  damaged 
corn  ;  if  this  be  true,  the  poisonous  substance  must  be  volatile. 

Clinicians  generally  state  that  pellagra  consists  of  three  stages, 
which  are  more  or  less  marked  and  distinct.  The  first  begins  with 
disturbances  of  the  digestive  organs,  the  tongue  is  heavily  coated, 
but  later  it  loses  its  epithelium,  there  is  loss  of  appetite  as  a  rule, 
although  in  exceptional  cases  the  desire  for  food  may  be  inordinate. 
Usually  there  is  diarrhea,  but  obstinate  constipation  may  occur. 
Accompanying  these  digestive  disturbances  there  is  pain  in  the  head, 


MAIDISMUS.  225 

neck  and  back.  Dizziness,  muscular  weakness  and  unsteadiness  of 
gait  are  frequently  observed.  Mental  activity  becomes  slow,  and 
some  complain  of  a  numbness  in  the  brain.  In  the  majority  of  in- 
stances, not  in  all,  there  is  a  characteristic  erythema  which  is  most 
marked  on  the  unclothed  parts  of  the  body,  as  the  hands  and  face, 
though  it  may  be  much  more  widely  distributed.  It  is  to  this  affec- 
tion of  the  skin  that  the  disease  owes  its  common  name,  pellagra 
(from  pelle,  skin,  and  agra,  rough). 

The  appearance  of  certain  cerebro-spinal  symptoms  characterizes 
the  second  stage  of  the  disease.  Chilly  sensations  are  complained 
of  and  there  is  often  constant  ringing  in  the  ears.  The  muscular 
weakness  is  increased  ;  tremors  and  convulsive  twitchings  become 
frequent,  and  cramps  and  light  spasms  occur.  The  tendon  reflex  is 
more  markedly  exaggerated,  sensibility  is  often  diminished  and  the 
patient  falls  into  a  state  of  melancholia.  The  skin  becomes  pale  or 
there  is  capillary  injection  over  certain  areas  notably  of  the  face, 
and  in  some  instances  the  skin  becomes  hard  and  scaly. 

Marked  emaciation  is  one  of  the  characteristic  symptoms  of  the 
third  stage.  The  subcutaneous  fat  wholly  disappears,  locomotion 
becomes  impossible,  incontinence  of  urine  is  persistent  and  uncon- 
trollable diarrhoea  makes  the  bed-ridden  patient  an  object  of  pity. 
Fortunately,  after  this  stage  has  been  reached  the  individual  loses  all 
resistance  to  the  infectious  diseases,  and  tuberculosis  or  septicemia 
frequently  closes  the  history.  A  considerable  number  of  pellagrous 
individuals  end  their  sufferings  by  suicide. 

The  most  characteristic  post-mortem  findings  may  be  stated  as 
follows  :  The  body  is  greatly  emaciated,  the  intestinal  walls  are  thin 
on  account  of  the  wasting  away  of  the  muscular  coat,  ulceration  in 
the  intestines  is  frequently  found,  the  cells  of  the  liver  and  of  the 
skin  and  the  muscles  of  the  heart  are  deeply  pigmented.  Atrophy 
seems  to  be  most  marked  in  those  organs  connected  with  the  vagus 
nerve,  the  lungs,  heart,  kidneys,  spleen  and  intestines.  Although 
marked  alterations  from  the  normal  are  frequently  found  in  the 
brain  and  cord,  there  seems  to  be  no  constant  or  characteristic  lesion 
in  these  organs. 


15 


CHAPTER    XL 

THE   EXAMINATION   OF   POISONOUS  FOODS. 

OuTBKEAKS  of  bromatotoxismus  have  become  common  in  recent 
years  and  chemists  and  bacteriologists  are  frequently  asked  to 
examine  foods  which  are  suspected  of  having  caused  untoward 
results.  The  increase  in  the  number  of  cases  of  this  kind  is  partly 
real  and  partly  only  apparent.  One  cause  of  the  actual  increase  lies 
in  the  larger  consumption  of  preserved  foods.  Meats,  the  appear- 
ance and  odor  of  which  would  render  their  sale  in  the  piece  im- 
possible, or  at  least  doubtful,  may  be  chopped,  cooked,  canned,  and 
sold  as  a  first-class  article.  We  do  not  state  that  this  fraud  is  com- 
monly practised,  but  that  it  is  a  possible  one  cannot  be  denied,  and 
that  it  is  occasionally  resorted  to  has  been  demonstrated  both  in  this 
country  and  in  Europe.  This  source  of  danger  to  the  public  health 
will  not  be  removed  until  the  necessity  for  scientific  inspection  of 
foods,  especially  of  animals  before  slaughtering,  is  understood  and 
practised.  However,  the  greater  number  of  cases  of  poisoning  by 
prepared  foods  arises  from  imperfections  in  methods  or  from  want  of 
intelligent  and  conscientious  attention  to  details.  When  we  recog- 
nize the  fact  that  the  successful  preparation  of  every  portion  of  pre- 
served food  depends  upon  the  exclusion  of  microorganisms,  both 
specific  and  putrefactive,  and  when  we  learn  that  the  processes  are 
carried  out  for  the  most  part  by  those  who  are  ignorant  of  the  scien- 
tific principles  involved,  then  we  can  only  wonder  that  the  health 
of  the  consumer  is  not  more  frequently  placed  in  jeopardy. 

The  apparent  increase  in  the  number  of  instances  of  food  poisoning 
is  due  to  the  fact  that  the  medical  profession  has  only  recently 
learned  to  recognize  food  infection  as  a  cause  of  illness  or  has  been 
in  possession  of  the  knowledge  necessary  to  convert  suspicion  into 
positive  demonstration.  Only  a  few  years  ago  we  were  seeking  for 
the  cause  of  cholera  infantum  in  mysterious  and  indefinite  telluric  or 
meteorological  conditions,  but  now  we  know  that  this  disease  is 
solely  due  to  infected,  and  consequently  poisonous,  food.  Formerly, 
many  of  these  cases  were  believed  to  be  due  to  the  accidental  or 
criminal  addition  of  some  metallic  or  vegetable  poison  to  the  food, 
and  unjust  accusation,  possibly  in  some  instances,  unjust  execution, 
resulted.  We  have  also  learned  that  typhoid  and  typhus  fevers, 
scarlet  fever,  and  other  acute  exanthemata,  and  even  pneumonia, 
may  be  closely  simulated  by  the  symptoms  due  to  infected  foods. 

Unfortunately,  the  expression  "  ptomain  poisoning  "  has  come  into 

226 


EXAMINATION  OF  POISONOUS  FOODS  227 

quite  general  use  to  designate  illness  due  to  infected  food.  While  it 
is  true  that  basic  substances  of  bacterial  origin  constitute  in  some 
instances  the  actual  materies  morbi,  this  is  not  always,  or  even  gen- 
erally, the  case.  Among  the  poisonous  bacterial  products  there  are 
many  that  are  not  basic,  and  many  others  concerning  the  chemical 
nature  of  which  we  are  yet  very  much  in  ignorance.  In  a  large 
proportion  of  the  instances  we  are  ignorant  not  only  of  the  chemistry 
of  the  poisons  w'hich  induce  the  untoward  effects,  but  of  the  bacteria 
through  the  activity  of  which  these  poisons  are  generated.  More- 
over, we  cannot  in  cases  of  bromatotosismus  draw  a  sharp  line  of 
distinction  between  intoxication  and  infection.  Food  poisoning  may 
originate  in  either  of  the  following  ways  :  (1)  The  food  is  infected 
and  the  poison  is  generated  only  and  wholly  before  the  food  is 
taken.  (2)  The  infecting  organism  may  begin  the  elaboration  of  its 
poisonous  products  outside  of  and  continue  the  same  process  inside 
the  body.  (3)  The  infection  may  not  result  in  the  production  of 
poisons  until  the  food  is  taken  into  the  body.  In  all  of  these  forms, 
infection  of  the  food  is  the  essential  element ;  it  is  this  that  must  be 
prevented,  and  to  this  especial  attention  must  be  called. 

How  shall  we  proceed  in  the  examination  of  food  suspected  of 
having  caused  sickness  or  death  ? 

In  the  first  place,  the  possibility  of  the  ill  effects  having  been  due 
to  metallic  poisons  should  be  considered.  In  cases  in  which  this 
possibility  exists  such  poisons  should  be  sought  by  methods  given  by 
the  best  toxicologists,  and  of  which  it  is  not  the  purpose  of  this  book 
to  speak.  In  case  the  substance  to  be  examined  consists  of  canned 
food  the  tests  for  mineral  poisons  should  always  be  made.  How- 
ever, when  a  teaspoonful  of  ice  cream  causes  nausea  and  vomiting, 
the  idea  that  these  effects  can  be  due  to  sulphate  of  zinc  dissolved  in 
the  freezer  is  too  preposterous  and  absurd  to  be  worthy  of  serious 
consideration  by  anyone  familiar  with  the  quantity  of  this  salt  neces- 
sary to  act  as  an  emetic. 

If  there  be  a  sufficient  quantity  of  the  food  a  portion  of  it  should 
be  fed  to  animals.  As  a  rule,  the  best  animals  for  experiments  of 
this  kind  are  puppies  and  kittens.  It  should  be  remembered  that 
rabbits  and  guinea-pigs  cannot  vomit  and  we  have  learned  by  experi- 
ments that  guinea-pigs  fed  exclusively  upon  pure  milk  die  ;  they  are 
not  able  to  digest  the  casein  which  forms  hard  balls  in  the  small  in- 
testines and  mushy  masses  in  the  large  intestines,  and  the  animals 
succumb  within  eight  or  ten  days,  apparently  from  intestinal  ob- 
struction. If  they  be  fed  upon  hay  or  other  vegetable  food  along 
with  the  milk,  they  are  apparently  able  to  digest  the  casein.  How- 
ever, under  no  circumstances  are  they  fit  animals  for  experimenta- 
tion, when  the  purpose  is  to  determine  whether  or  not  milk  or  any 
of  its  products  is  poisonous. 

A  thorough  examination  of  foods  for  bacterial  poisons  cannot  be 


228  EXAMINATION  OF  POISONOUS  FOODS. 

made  except  in  a  properly  equipped  laboratory.  It  is  our  purpose 
to  briefly  point  out  the  methods  that  may  be  followed,  and  in  doing 
so  we  take  it  for  granted  that  the  one  who  attempts  work  of  this 
kind  is  already  familiar  with  the  ordinary  technic  of  bacteriological 
research.  The  line  of  procedure  will  vary  somewhat  with  the  kind 
of  food  to  be  examined,  the  form  in  which  it  has  been  prepared,  and 
the  quantity  supplied  the  analyst.  All  samples  should  be  examined 
with  as  little  delay  as  possible  after  the  article  has  become  the  object 
of  suspicion.  When  delay  is  unavoidable,  farther  bacterial  growth 
should  be  retarded  in  the  meantime  as  far  as  possible ;  not  by  anti- 
septics, but  by  low  temperature.  Microorganisms  not  present  at  the 
time  of  the  supposed  poisoning  may  be  accidentally  introduced,  or 
non-toxicogenic  bacteria  may  multiply  to  such  an  extent  that  the 
detection  of  the  harmful  organism  is  rendered  impossible. 

As  a  rule,  the  quantity  of  the  food  supplied  the  analyst  is  not 
sufficient  to  allow  of  the  detection  or  the  isolation  of  the  chemical 
poison  directly.  To  try  to  find  the  toxin  in  a  few  ounces  of  cheese 
or  a  small  bit  of  meat  by  direct  extraction,  is  a  task  that  would  be 
undertaken  only  by  one  quite  ignorant  of  the  nature  of  these  poisons. 
In  all  but  exceptional  instances,  where  many  pounds  of  food  are 
supplied,  the  portion  that  reaches  the  laboratory  can  only  be  re- 
garded as  the  bearer  of  the  germ  to  the  activity  of  which  the  poison 
is  due.  This  germ  must  be  detected,  isolated,  grown  in  pure  cul- 
tures, and  its  toxicogenic  properties  demonstrated  upon  the  lower 
animals.  It  should  be  clearly  understood  that  the  most  thorough 
study  of  the  morphological  characteristics  of  the  germ  and  of  the 
chemical  properties  of  the  poison  will  not  suffice  without  an 
accompanying  determination  of  the  toxicological  action  of  the 
culture.  The  infectious  nature  of  the  bacterium  should  also  be 
studied. 

It  should  always  be  borne  in  mind  that  the  article  of  food  has 
probably  been  through  several  hands,  some  of  which  may  not  have 
been  germ-free.  In  the  examination  of  pieces  of  meat  and  cheese, 
the  surface  should  be  sterilized  with  a  broad,  heated  knife  or  other 
piece  of  iron.  It  has  been  shown  that  bacteria  deposited  on  such 
surfaces  penetrate  slowly.  Then  with  other  sterilized  knives,  sec- 
tions should  be  made,  and  one  or  more  small  bits  taken  from  the  in- 
terior should  be  placed  in  sterilized  bouillon,  milk  or  other  culture 
medium.  Not  less  than  a  dozen  tubes  should  be  inoculated  in  this 
way.  Three  of  these  should  be  grown  aerobically  at  ordinary  tem- 
perature ;  three  anaerobically  at  the  same  temperature ;  three  aero- 
bically at  37°  and  three  anaerobically  at  37°.  Some  of  the  toxico- 
genic germs  grow  best  at  relatively  low  temperature  (20°  to  25°) 
and  fail  to  develop  at  37°.  Others  have  their  optimum  growth  at 
the  last  mentioned  temperature.  Some  develop  only  when  the  air  is 
excluded,  and  others  only  when  freely  supplied  with  air. 


EXAMINATION  OF  POISONOUS  FOODS.  229 

In  the  examination  of  liquid  and  semi-liquid  foods,  such  as  milk, 
custard,  cream,  broths,  and  jellies,  small  bits  or  a  few  drops  should 
be  placed  in  sterilized  media  and  grown  under  the  conditions  above 
mentioned. 

A  growth  having  occurred  in  one  or  more  of  these  tubes,  the  bac- 
teria should  be  examined  in  hanging-drops  and  in  stained  mounts. 
If  more  than  one  organism  be  present,  plate  cultures  should  be  made 
and  each  germ  should  again  be  grown  under  the  conditions  men- 
tioned. 

The  infectious  character  of  each  organism  should  be  tested  on  the 
lower  animals  :  (1)  By  feeding;  (2)  by  subcutaneous  inoculation; 
(3)  by  intra-peritoneal  inoculation,  and  (4)  by  intravenous  injection. 
The  animals  generally  employed  in  these  experiments  are  white  mice, 
white  rats,  guinea-pigs,  kittens,  puppies,  and  rabbits.  A  given  germ 
may  be  toxicogenic  to  one  of  these  animals  and  not  to  the  others. 
The  quantity  of  the  bouillon  culture — twenty-four  hours  old  or  older 
— employed  should  be  relatively  large — from  one  to  ten  c.c,  accord- 
ing to  the  animal  and  the  method  of  inoculation.  If  these  amounts 
prove  active,  smaller  quantities  should  be  tried  until  the  limit  is 
reached. 

Cultures  sterilized  both  by  filtration  and  by  heat  should  be  tested 
on  animals.  It  should  be  borne  in  mind  that  certain  toxins,  notably 
those  of  the  colon  bacilli,  are  removed  from  cultures  by  filtration 
through  porcelain,  while  they  are  not  altered  by  a  degree  of  heat 
sufficient  to  kill  the  bacteria. 

If  by  the  above  mentioned  experiments  a  toxicogenic  germ  has  been 
discovered,  its  morphological,  cultural,  tinctorial  and  pathogenic 
properties  may  be  studied  as  thoroughly  as  the  investigator  may  desire. 
The  study  of  the  bacterial  poison  may  also  be  carried  to  the  same 
extent.  The  examination  for  ptomains  and  toxins  can  be  carried  out 
according  to  the  methods  described  in  subsequent  chapters. 


CHAPTER  XII. 

METHODS   OF   EXTRACTING  PTOMAINS. 

From  what  has  been  given  in  the  preceding  pages,  one  may  form 
an  idea  of  the  diifficulties  with  which  the  chemist  has  to  contend  in 
endeavoring  to  isolate  the  basic  products  of  bacterial  growth.  He 
has  to  deal  with  complex  substances,  the  nature  and  reactions  of 
many  of  which  he  does  not  know.  Moreover,  the  bodifes  which  he 
seeks  to  isolate  are  often  prone  to  undergo  decomposition  and  in  this 
way  escape  detection.  Many  ptomains  are  volatile  or  decomposable 
at  temperatures  near  that  of  boiling  water,  and  in  such  cases  so- 
lutions cannot  be  evaporated  in  the  ordinary  way  and  the  poison 
remain  in  the  residue.  The  investigator  has  frequently  been  disap- 
pointed when  on  the  evaporation  of  a  solution,  which  he  has  demon- 
strated to  be  poisonous,  he  j&nds  that  the  residue  is  wholly  inert. 
Again,  he  may  destroy  the  substance  which  he  attempts  to  isolate 
with  the  reagents  which  he  employs.  So  simple  a  procedure  as  the 
removal  of  a  metallic  base  from  a  solution  containing  a  ptomain,  by 
precipitation  with  hydrogen  sulphid,  has  been  known  to  wholly  de- 
stroy the  ptomain.  Probably  the  most  perplexing  difficulty  in  the 
isolation  of  these  putrefactive  alkaloids  lies  in  the  great  number, 
complexity,  and  diversity  of  the  other  substances  present  in  the  decom- 
posing matters.  The  same  ptomain  may  be  present  in  equal  quan- 
tities in  two  samples  of  milk,  and  yet  it  may  be  easily  obtained  from 
the  one,  while  from  the  other  only  minute  traces  can  be  secured. 
The  difference  is  due  to  the  fact  that  the  other  constituents  of  the 
milk  in  the  two  samples  are  at  different  stages  of  the  putrefactive 
process,  and,  consequently,  differ  in  their  reactions  and  in  their 
effects  upon  the  agents  employed  to  isolate  the  poison.  All  chem- 
ists appreciate  this  difficulty. 

The  first  thing  for  the  chemist,  who  undertakes  this  work,  to  do  is 
to  ascertain  whether  or  not  his  reagents  are  pure.  We  have  found 
samples  of  German  ether,  imported  on  account  of  its  supposed 
purity,  to  yield  on  spontaneous  evaporation  a  residue  which  gave 
some  of  the  alkaloidal  reactions,  and  a  few  drops  of  which,  injected 
under  the  skin  of  a  frog,  caused  paralysis  and  death  within  a  few 
minutes.  We  advise  that  500  c.c.  of  the  ether  to  be  used  be 
allowed  to  evaporate  spontaneously,  and  its  residue,  if  there  be 
one,  be  examined  both  chemically  and  physiologically.  The 
basic  substance  which  exists  in  some  samples  of  sulphuric  ether  is 
pyridin. 

230 


THE  STAS-OTTO  METHOD.  231 

Guareschi  and  Mosso  found  commercial  alcohol  almost  invariably 
to  contain  small  quantities  of  an  alkaloidal  substance,  the  odor  of 
which  is  similar  to  that  of  nicotin  and  pyridin.  Its  solutions  are 
precipitated  by  the  general  alkaloidal  reagents,  with  the  exception 
of  platinum  chlorid  and  tannic  acid.  It  does  not  reduce,  or  reduces 
feebly,  ferric  salts.  From  one  sample  of  alcohol  they  obtained  a 
base  which,  on  addition  to  the  above  reagents,  did  give  a  precipitate 
with  platinum  chlorid.  Alcohol  may  be  freed  from  these  substances 
by  distillation  over  tartaric  acid. 

In  amylic  alcohol,  Haitinger  found  almost  0.5  per  cent,  of  pyridin. 
This  reagent  may  be  purified  in  the  manner  recommended  for 
ethylic  alcohol. 

Chloroform,  when  found  to  leave  a  residue  on  evaporation,  should 
be  washed  first  with  distilled  water,  then  with  distilled  water  ren- 
dered alkaline  with  potassium  carbonate,  then  dried  over  calcium 
chlorid  and  distilled. 

Petroleum  ether  sometimes  contains  a  base  which  has  an  odor 
similar  to  trimethylamin  or  pyridin,  and  which  gives  a  precipitate 
with  platinum  chlorid,  forming  in  octahedra  ;  benzol  may  contain  a 
similar  substance. 

The  following  methods  have  been  used  for  the  purpose  of  extract- 
ing putrefactive  alkaloids  : 

The  Stas-Otto  Method. — This  method  depends  upon  the  follow- 
ing facts :  (1)  The  salts  of  the  alkaloids  are  soluble  in  water  and 
alcohol,  and  generally  insoluble  in  ether,  and  (2)  the  free  alkaloids 
are  soluble  in  ether,  and  are  removed  from  alkaline  fluids  by  agita- 
tion with  ether.  These  principles  are  capable  of  great  variations  in 
their  application.  The  usual  directions  are  as  follows  :  Treat  the 
mass  under  examination  with  about  twice  its  weight  of  90  per  cent, 
alcohol,  and  from  ten  to  thirty  grains  of  tartaric  or  oxalic  acid  ;  digest 
the  whole  for  some  time  at  about  70°,  and  filter.  Evaporate  the 
filtrate  at  a  temperature  not  exceeding  35°  either  in  a  strong  current 
of  air  or  in  vacuo  over  sulphuric  acid.  Take  up  the  residue  with 
absolute  alcohol,  filter,  and  again  evaporate  at  a  low  temperature. 
Dissolve  this  residue  in  water,  render  alkaline  with  sodium  carbon- 
ate, and  agitate  with  ether.  After  separation  remove  the  ether  with 
a  pipette,  or  by  means  of  a  separator,  and  allow  it  to  evaporate  spon- 
taneously. The  residue  may  be  further  purified  by  redissolving  in 
water,  and  again  extracting  with  ether. 

The  following  modifications  of  this  method  are  employed  : 

Instead  of  tartaric  or  oxalic  acid,  acetic  acid  may  be  used. 

When  the  fluid  suspected  of  containing  a  ptomai'n  is  already  acid 
from  the  development  of  lactic  or  other  organic  acid,  the  addition  of 
an  acid  may  be  dispensed  with. 

Ether  extracts  are  made  from  both  acid  and  alkaline  solutions. 


232 


METHODS  OF  EXTRACTING  PTOMAINS. 


Chloroform,  amylic  alcohol,  and  benzene  are  used  as  solvents  after 
extraction  with  ether. 


Dragendorffs  Method. — The  finely  divided  substance  is  digested 
for  some  hours  with  water  acidulated  with  sulphuric  acid,  at  from 
40°  to  50°.  This  is  repeated  two  or  three  times,  and  the  united 
filtered  extracts  are  evaporated  to  a  syrup,  which  is  treated  with 
four  volumes  of  alcohol  and  digested  for  twenty-four  hours  at  30°. 
After  cooling,  the  alcoholic  extract  is  filtered,  the  residue  washed 
with  70  per  cent,  alcohol,  and  the  united  filtrates  freed  from  alcohol 
by  distillation.  The  aqueous  residue,  diluted  if  desirable,  is  filtered 
and  submitted  to  the  following  extractions  : 

1.  The  acid  liquid  is  shaken  with  freshly  rectified  petroleum  ether, 
as  long  as  this  reagent  leaves  a  residue  on  evaporation. 

2.  The  acid  fluid  is  now  extracted  with  benzene. 

3.  The  next  solvent  used  is  chloroform. 

4.  The  liquid  is  now  again  extracted  with  petroleum  ether  in  order 
to  remove  traces  of  benzene  and  chloroform. 

5.  The  liquid  is  next  made  alkaline  with  ammonia  and  succes- 
sively extracted  with  petroleum  ether,  benzene,  chloroform,  and  amy- 
lic alcohol. 

6.  The  remainder  of  the  ammoniacal  liquid  is  mixed  with  pow- 
dered glass,  evaporated  to  dryness,  the  residue  pulverized,  and  ex- 
tracted with  chloroform. 

The  residue  obtained  with  each  of  the  above  solvents  should  be 
examined  for  ptomains. 

Brieger's  Method. — The  substance  under  examination  is  divided 
as  finely  as  possible,  and  then  heated  with  water  slightly  acidified 
with  hydrochloric  acid.  During  the  heating  care  must  be  taken  that 
the  feebly  acid  reaction  is  maintained,  and  the 
heat  should  continue  for  only  a  few  minutes. 
The  liquid  is  then  filtered  and  concentrated,  at 
first  on  a  plate  and  then  on  the  water-bath,  to 
a  syrup.  If  one  has  highly  odorous  material,  a 
piece  of  apparatus  devised  by  Bocklisch  is  of 
service.  The  fluid  to  be  evaporated  is  placed  in 
a  globular  flask,  the  rubber  stopper  of  which 
carries  two  small  glass  tubes,  one  of  which,  B, 
extends  to  the  bottom  of  the  flask,  while  A  is 
connected  with  a  water -pump  or  aspirator,  which 
draws  the  vapor  through  the  tube.  In  order  to 
prevent  the  return  of  condensed  fluids,  the  end 
of  A  in  the  flask  is  curved  on  itself.  The  tube  B 
is  finely  drawn  out  and  through  it  a  current 
of  air  is   constantly   moving  and  prevents  the 


Fig.  2. 


THE  METHODS  OF  GAUTIEB  AND  ETARD.  233 

formation  of  a  deposit  or  a  pellicle  in  the  fluid.  By  regulating 
the  amount  of  air  entering  through  this  tube,  more  or  less  of 
a  vacuum  will  be  formed  in  the  flask.  After  evaporation  to  a  syrup, 
an  extraction  is  made  with  96  per  cent,  alcohol  and  the  filtered  ex- 
tract is  treated  with  a  warm  alcoholic  solution  of  lead  acetate.  The 
lead  precipitate  is  removed  by  filtration,  the  filtrate  evaporated  to  a 
syrup  and  again  extracted  with  96  per  cent,  alcohol.  The  alcohol 
is  driven  off";  the  residue  taken  up  with  water;  traces  of  lead  re- 
moved with  hydrogen  sulphid  ;  and  the  filtrate,  acidified  with  hy- 
drochloric acid,  evaporated  to  a  syrup,  which  is  extracted  with  alco- 
hol, and  the  filtrate  precipitated  with  an  alcoholic  solution  of  mer- 
curic chlorid.  The  mercury  precipitate  is  boiled  with  water,  and  on 
account  of  the  difierences  in  solubility  of  the  double  compounds  with 
mercury,  one  ptomain  may  be  separated  from  others  at  this  stage  of 
the  process.  (If  thought  best,  the  lead  precipitate  may  be  freed 
from  lead  and  carried  through  the  subsequent  steps  of  the  process. 
Brieger  found  small  quantities  of  ptomains  in  the  lead  precipitate 
only  in  his  work  with  poisonous  mussels.) 

The  mercury  filtrate  is  freed  from  mercury,  evaporated,  and  the 
excess  of  hydrochloric  acid  carefully  neutralized  with  soda  (the  re- 
action is  kept  feebly  acid)  ;  then  it  is  again  taken  up  with  alcohol  in 
order  to  free  it  from  inorganic  salts.  The  alcohol  is  evaporated, 
the  residue  taken  up  with  water,  the  remaining  traces  of  hydrochloric 
acid  neutralized  with  soda,  the  whole  acidified  with  nitric  acid,  and 
treated  with  phosphomolybdic  acid.  The  phosphomolybdate  double 
compound  is  separated  by  filtration  and  decomposed  with  neutral 
acetate  of  lead.  This  is  hastened  by  heating  on  the  water-bath. 
The  lead  is  removed  by  hydrogen  sulphid,  the  filtrate  is  evaporated 
to  a  syrup  and  taken  up  with  alcohol,  from  which  many  ptomains 
are  deposited  as  chlorids,  or  double  salts  may  be  formed  in  the 
alcoholic  solution.  The  chlorids  as  deposited  from  the  alcoholic  solu- 
tion are  seldom  pure  and  may  be  isolated  by  precipitation  with  gold 
chlorid,  platinum  chlorid,  or  picric  acid,  and  on  account  of  the  dif- 
ference in  solubility  of  these  double  salts,  the  process  of  purification 
is  rendered  more  easy.  The  chlorid  of  the  base  is  obtained  by  re- 
moving the  metal  with  hydrogen  sulphid,  while  the  picrate  is  taken 
up  with  water,  acidified  with  hydrochloric  acid,  and  repeatedly  ex- 
tracted with  ether,  in  order  to  remove  the  picric  acid. 

The  Methods  of  Gautier  and  Etard. — The  putrid  matters, 
liquid  and  solid,  are  distilled  at  a  low  temperature  in  vacuo.  The 
distillate  (A)  may  contain  ammonium  carbonate,  phenol,  skatol, 
trimethylamin,  and  the  volatile  fatty  acids.  The  residue  after  fil- 
tration is  treated  successively  by  ether  and  alcohol. 

The  extraction  with  ether  {B)  separates  the  ptomain  and  some 
fatty  acids.     The  alcoholic  extract  (C)  removes  the  remainder  of  the 


234  METHODS  OF  EXTRACTING  PTOMAINS. 

fatty  acids,  as  well  as  the  acid  and  neutral  nitrogenous  bodies,  al- 
most all  of  which  are  crystallizable.  The  insoluble  residue  is  boiled 
with  dilute  hydrochloric  acid,  with  exclusion  of  air,  finally  evapor- 
ated to  dryness,  and  the  residue  again  extracted  with  alcohol.  This 
nearly  alkaline  solution  (JJ)  can  be  divided  by  acetate  and  subace- 
tate  of  lead  into  two  portions. 

Gautier  has  also  employed  the  following  method  :  The  putrid 
liquids,  after  the  removal  of  fats,  are  feebly  acidified  with  dilute 
sulphuric  acid,  then  distilled  in  vacuo  at  a  low  temperature.  The 
distillate  contains  ammonia,  phenol,  indol,  and  skatol.  The  syrupy 
residue,  separated  from  any  crystals  which  may  have  formed,  is 
rendered  alkaline  with  baryta,  filtered,  and  extracted  a  number  of 
times  with  chloroform,  in  order  to  dissolve  the  bases.  The  solution 
is  distilled  at  a  low  temperature,  either  in  vacuo  or  in  a  current  of 
carbonic  acid.  The  contents  of  the  retort,  on  being  treated  with 
water  and  tartaric  acid,  separate  into  a  brown  resin  and  a  liquid 
portion.  The  latter  is  removed  and  treated  with  a  dilute  solution 
of  potash,  when  it  gives  off  the  odor  of  carbylamin,  which  was  dis- 
covered by  Gautier  in  1866,  and  which  according  to  Calmel,  is  a 
constituent  of  the  venom  of  toads.  The  alkali  also  sets  free  the 
bases,  which  are  removed  by  extraction  with  ether,  and  the  ether 
evaporated  in  a  current  of  carbonic  acid  under  slight  pressure,  then 
under  a  bell-jar  over  caustic  potash.  The  bases  may  be  separated 
by  fractional  precipitation  with  platinum  chlorid,  or,  if  present  in 
sufficient  quantity,  by  distillation  in  vacuo. 

In  some  instances,  Gautier  modified  his  method  as  follows  :  The 
alkaline  putrid  liquid  is  treated  with  oxalic  acid  to  free  acidulation, 
and  as  long  as  the  fatty  acids  continue  to  separate.  The  liquid  is 
then  warmed  and  distilled  until  the  distillate  ceases  to  be  turbid. 
Pyrrol,  skatol,  phenol,  indol,  volatile  fatty  acids,  and  some  of  the 
ammonia  pass  over.  The  portion  remaining  in  the  retort  is  rendered 
alkaline  with  lime  water  and  the  precipitate  which  forms  and  con- 
tains the  greater  part  of  the  fixed  acids  is  removed.  The  liquid 
portion,  which  is  alkaline,  is  distilled  to  dryness,  care  being  taken 
to  receive  the  distillate  in  dilute  sulphuric  acid.  The  bases  and 
ammonia  pass  over.  The  distillate  is  neutralized  with  sulphuric  acid 
and  evaporated  almost  to  dryness,  then  decanted  from  the  ammonium 
sulphate  which  crystallizes.  The  mother  liquor  is  extracted  with 
concentrated  alcohol,  which  dissolves  the  sulphates  of  the  ptomains. 
After  driving  ofi"  the  alcohol  the  residue  is  rendered  alkaline  with 
caustic  soda,  and  successively  extracted  with  ether,  petroleum  ether, 
and  chloroform.  The  lime  precipitate  is  dried  and  extracted  with 
ether  which  removes  any  fixed  bases  that  may  be  present. 

Remarks  Upon  the  Methods. — Guareschi  and  Mosso  condemn 
the  method  of  Dragendorff,  inasmuch  as  they  have  found  that  basic 


REMARKS  UPON  THE  METHODS.  235 

bodies  are  formed  by  the  action  of  dilute  sulphuric  acid  upon  unde- 
composed  albuminous  substances,  and  they  recommend  the  employ- 
ment of  the  Stas-Otto  method  with  these  conditions :  (1)  No  more 
acid  should  be  added  than  is  absolutely  necessary  to  keep  the  reac- 
tion acid ;  (2)  the  heat  used  in  evaporation  should  not  be  great,  and 
it  is  better  that  evaporation  should  be  made  in  vacuo.  In  this  way, 
they  say,  no  basic  substance  will  be  obtained  from  fresh  tissue. 

Marino-Zuco  ascertained  that  by  treating  fresh  eggs,  brain,  liver, 
spleen,  kidney,  lungs,  heart  and  blood  by  either  the  Stas-Otto  or  the 
Dragendorff  method,  he  could  obtain  a  substance  which  gave  alka- 
loidal  reactions,  and  which  he  demonstrated  to  be  cholin.  His  ex- 
periments led  him  to  believe  that  cholin  does  not  exist  preformed  in 
these  fresh  tissues,  but  that  it  results  from  the  action  of  the  dilute 
acids  upon  lecithin.  Cholin  was  found  most  abundantly  in  those 
tissues  which  are  rich  m  lecithin,  such  as  the  yolks  of  eggs,  brain, 
liver  and  blood  ;  while  only  traces  could  be  obtained  from  the  whites 
of  eggs,  lungs  and  the  heart.  The  method  of  Dragendorff*  was  found 
to  furnish  much  larger  quantities  of  cholin  than  could  be  obtained  by 
the  Stas-Otto  process.  Coppola  agrees  with  his  countrymen  in  con- 
demning the  method  of  Dragendorff"  as  a  means  of  extracting  pto- 
mai'ns.  However,  the  Stas-Otto  method  is  by  no  means  perfect,  and 
the  principal  difficulties  met  with  in  its  application  are  as  follows  : 
(1)  In  most  instances  the  extraction  of  the  base  is  incomplete ;  (2) 
the  degree  to  which  the  putrefactive  alkaloid  is  removed  by  the  sol- 
vent depends  largely  upon  the  nature  of  the  other  substances  pres- 
ent. This  fact  in  some  cases  aids  and  in  others  hinders  the  labors 
of  the  investigator ;  thus,  several  ptomains,  which  when  pure  are 
wholly  insoluble  in  ether,  may  be  removed  in  part,  at  least,  from  or- 
ganic mixtures  by  this  solvent  by  passing  into  the  solution  along 
with  other  substances  ;  but  if  the  attempt  be  made  to  purify  one  of 
these  bases  by  repeated  solution  and  extraction  with  ether,  the  re- 
sult is  failure,  because  the  more  completely  the  alkaloid  is  freed  from 
impurities,  the  less  soluble  in  ether  it  becomes.  This  criticism  is 
equally  applicable  to  the  Dragendorff"  method,  and  to  all  others  in  so 
far  as  extractions  are  made.  However,  we  may  state  that  the  Stas- 
Otto  method  is  in  suitable  cases  the  best  that  can  be  employed.  By  it 
the  substances  are  submitted  to  the  least  chemical  manipulation,  and 
the  results  obtained  are  the  most  reliable.  Many  of  the  more  com- 
plex putrefactive  products  are  so  easily  decomposed  or  otherwise  al- 
tered that  the  investigator  should  seek  to  isolate  them  by  the  sim- 
plest methods  possible.  If  it  can  be  done  without  the  addition  of  any 
acid  or  without  the  application  of  heat,  so  much  the  better. 

By  his  method,  Brieger  discovered  a  number  of  basic  bodies,  and 
gave  great  impetus  to  the  study  of  the  chemistry  of  putrefaction  ; 
but  it  is  open  to  the  objection  that  basic  substances  may  be  formed 
by  the  action  of  the  reagents  used,  and  Gram  states  that  when  the 


236  METHODS  OF  EXTRACTING  PTOMAINS. 

lactate  of  cholin,  an  inert  substance  which  is  widely  distributed  both 
in  plants  and  animals,  is  heated,  it  is  converted  into  a  poison  which 
has  a  muscarin-like  action.  However,  Brieger  obtained  some  of 
his  bases  by  a  much  simplified  modification  of  his  complete  method  ; 
for  instance,  in  obtaining  neuridin,  he  treated  the  aqueous  extract  of 
the  putrid  material,  after  boiling  and  filtration,  with  mercuric  chlorid, 
collected  the  precipitate,  decomposed  it  with  hydrogen  sulphid,  evap- 
orated the  filtrate  on  the  water-bath,  and  extracted  the  base  from  the 
residue  with  dilute  alcohol.  Brieger's  method  is  only  a  modification 
of  that  employed  by  Bergmann  and  Schmiedeberg  as  long  ago  as 
1868  in  the  preparation  of  sepsin  from  putrid  yeast. 


CHAPTER  XIII. 

THE  IMPORTANCE   OF  BACTERIAL  PRODUCTS  TO   THE 
TOXICOLOGIST. 

The  presence  in  the  cadaver  of  substances  which  give  not  only 
the  general  alkaloidal  reactions,  but  respond  to  some  of  the  tests 
which  have  hitherto  been  considered  characteristic  of  individual 
vegetable  alkaloids,  is  of  importance  to  the  toxicologist.  The  possi- 
bility of  mistaking  putrefactive  for  vegetable  alkaloids  should  always 
be  borne  in  mind  by  the  chemist  in  making  any  medico-legal  inves- 
tigations. On  the  other  hand,  cases  of  poisoning  with  bacterial  prod- 
ucts sometimes  terminate  fatally,  and  in  such  instances  the  chemist 
should  not  be  satisfied  with  determining  the  absence  of  mineral  and 
vegetable  poison,  but  should  strive  to  detect  in  the  food  or  in  the 
dead  body  positive  evidence  of  the  presence  of  the  putrefactive 
poison. 

We  herewith  append  a  brief  account  of  some  of  the  cases  in  which 
putrefactive  substances  have  been  found  to  resemble  in  their  reactions 
the  vegetable  alkaloids. 

Goniin-like  Substances. — In  the  Brandes-Krebs  trial,  which 
took  place  in  Braunschweig  in  1874,  two  chemists  obtained  from 
the  undecomposed  parts  of  the  body,  in  addition  to  arsenic,  an  alka- 
loid which  they  pronounced  coniin.  This  substance  was  referred  to 
Otto  for  further  examination  and  he  reported  that  it  was  neither 
coniin  nor  nicotin,  nor  any  vegetable  alkaloid  with  which  he  was 
acquainted.  He  converted  the  substance  into  an  oxalate,  dissolved 
it  in  alcohol,  evaporated  the  alcohol,  dissolved  the  residue  in  water, 
rendered  this  solution  alkaline  with  potash,  then  extracted  the  bases 
with  petroleum  ether.  On  evaporation  of  this  extract  the  alkaloid 
appeared  as  a  bright  yellow  oil,  which  had  a  strong  unpleasant  odor, 
quite  different,  however,  from  that  of  coniin.  It  was  strongly  alka- 
line, had  an  intensely  bitter  taste,  and  was  volatile  at  ordinary  tem- 
perature. From  its  aqueous  solution  it  was  precipitated  by  the 
chlorides  of  gold,  platinum  and  mercury.  In  these  reactions  it  re- 
sembled nicotin  from  which  it  differed  in  the  double  refractive  and 
crystalline  character  of  its  hydrochlorid.  With  an  ethereal  solution 
of  iodin  this  substance  did  not  give  the  Roussin  test  for  nicotin,  but 
instead  of  the  long  ruby-red  crystals  there  appeared  small,  dark  green, 
needle-shaped  ones. 

237 


238  IMPORTANCE  TO   TOXICOLOGIST. 

This  substance  was  found  to  be  highly  poisonous,  inasmuch  as  7 
eg.  injected  subcutaneously  into  a  large  frog  produced  instantaneous 
death,  and  44  mg.  given  to  a  pigeon  caused  a  similar  result.  On 
account  of  its  poisonous  properties  the  jury  of  medical  experts  de- 
cided that  the  substance  was  a  vegetable  alkaloid,  notwithstanding 
the  fact  that  Otto's  experiments  demonstrated  that  this  could  not  be 
true. 

Brouardel  and  Boutmy  found  in  the  body  of  a  woman  who  had 
died,  after  suflFering  with  ten  other  persons,  from  choleraic  symp- 
toms after  eating  of  a  stuffed  goose,  a  base  which  gave  the  odor  of 
coniin  and  the  same  reactions  with  gold  chlorid  and  iodin  in  potassium 
iodid,  etc.,  as  coniin.  The  same  base  was  found  in  the  remainder 
of  the  goose.  It  did  not  give  a  red  coloration  with  the  vapor  of 
hydrochloric  acid,  and  it  did  not  form  butyric  acid  on  oxidation, 
and,  although  it  was  poisonous,  it  did  not  induce  in  frogs  the 
symptoms  of  coniin  poisoning. 

Selmi  repeatedly  found  coniin-like  substances  in  decomposing 
animal  tissue.  By  distilling  an  alcoholic  extract  from  a  cadaver, 
acidifying  the  distillate  with  hydrochloric  acid,  evaporating,  treating 
the  residue  with  barium  hydrate  and  ether,  and  allowing  the  ether  to 
evaporate  spontaneously,  he  obtained  a  residue  of  volatile  bases,  the 
greater  portion  of  which  consisted  of  trimethylamin.  After  remov- 
ing the  trimethylamin  the  residue  had  the  odor  of  mouse  urine. 
Later,  Selmi  obtained  an  unmistakable  coniin  odor  from  a  chloro- 
form extract  of  the  viscera  of  a  person  who  had  been  buried  six 
months,  and  in  another  case  ten  months  after  burial.  Two  or  three 
drops  of  an  aqueous  solution  of  the  alkaline  residue  of  the  chloro- 
form extract,  allowed  to  evaporate  on  a  glass  plate,  gave  off"  such 
a  penetrating  odor  that  Selmi  was  compelled  to  withdraw  from  close 
proximity  to  the  substance,  and  the  odor  imparted  to  his  hands 
in  testing  with  the  general  alkaloidal  reagents  remained  for  half  an 
hour. 

An  aqueous  solution  of  a  ptomain  obtained  by  Selmi  by  extraction 
with  ether,  according  to  the  Stas-Otto  method,  from  the  unde- 
composed  parts  of  a  cadaver,  had  no  marked  odor,  but  after  being 
kept  for  a  long  time  in  a  sealed  tube  it  not  only  gave  off  a  coniin 
odor,  but  the  vapor  turned  red  litmus  paper  blue.  Again,  the  sul- 
phate of  a  ptomain  obtained  from  putrid  egg-albumin,  on  standing, 
formed  in  two  layers,  one  of  which  was  a  golden-yellow  liquid,  which 
on  treatment  with  barium  hydrate  gave  off  ammonia  and  later  the  odor 
of  coniin.  Since  butyric  and  acetic  acids  were  formed  by  the  oxida- 
tion of  this  base,  Selmi  concluded  that  it  was  real  coniin  or  methyl- 
coniin,  and  that  it  was  formed  by  the  oxidation  of  certain  fixed 
ptomains,  or  by  the  action  of  different  amido  bases  on  volatile  fatty 
acids.  The  substance,  which  was  found  by  Sonnenschein  in  a 
criminal  trial    in  East  Prussia  and   which  was  believed  by  that 


STRYCHNIN-LIKE  SUBSTANCES.  239 

chemist  to  be  the  alkaloid  of  the  water  hemlock  (Cicuta  virosa),  is 
thought  by  Otto,  Husemann,  and  others,  to  be  a  cadaveric  coniin. 
Otto  says  that  the  symptoms  reported  in  the  case  were  not  those  of 
either  coniin  or  cicuta.  Sonnenschein  obtained  the  base  six  weeks 
after  the  exhumation  of  the  body,  which  had  been  buried  three 
months.  The  base  had  the  odor  of  coniin,  the  taste  of  tobacco,  gave 
with  potassium  bichromate  and  sulphuric  acid  the  odor  of  butyric 
acid,  and  behaved  with  the  reagents  like  coniin. 

Husemann  states  that  it  is  very  difficult,  if  not  impossible,  for  the 
chemist  to  affirm  with  certainty  that  he  has  detected  true  coniin  in 
the  body.  The  symptoms  and  the  post-mortem  appearances  must 
conform  with  those  induced  by  the  vegetable  alkaloid.  The  analysis 
must  be  made  before  decomposition  sets  in,  and  the  amount  of  the 
base  found  must  be  sufficient  for  physiological  experimentation. 

A  Nicotin-like  Substance. — Wolckenhaar  obtained  from  the 
decomposed  intestines  of  a  woman,  who  had  been  dead  six  weeks,  by 
extraction  with  ether  from  an  alkaline  solution,  a  base  bearing  a 
close  resemblance  to  nicotin.  This  base  was  fluid,  at  first  yellow, 
but  becoming  brownish-yellow  on  exposure  to  the  air.  It  was 
strongly  alkaline  and  gave  off  an  odor  resembling  that  of  nicotin. 
but  stronger,  more  ethereal,  benumbing,  and  similar  to  that  of  fresh 
poppy  heads.  ,  It  was  soluble  in  all  proportions  of  water,  and  the  solu- 
tions, which  did  not  become  cloudy  on  the  application  of  heat,  did 
not  taste  bitter,  but  were  slightly  pungent.  The  peculiar  odor  did 
not  disappear  on  saturating  the  base  with  oxalic  acid.  The  hydro- 
chlorid  was  yellow  like  varnish,  had  a  strong  odor  and  became  moist 
on  exposure  to  the  air.  Under  the  microscope  it  showed  no  crystals, 
thus  differing  from  nicotin  hydrochlorid.  It  also  differed  from 
nicotin  in  its  reactions  with  potassio-bismuthic  iodid,  gold  chlorid, 
iodin  solution,  mercuric  chlorid,  and  platinum  chlorid,  and  it  did 
not  give  the  Eoussin  test.  Furthermore,  it  could  not  be  identified 
with  trimethylamin,  spartein,  mercurialin,  lobelin,  or  other  fluid  and 
volatile  bases. 

Strychnin-like  Substances. — In  a  criminal  prosecution  at  Verona, 
Ciotta  obtained  from  the  exhumed,  but  only  slightly  decomposed, 
body,  an  alkaloid  which  gave  a  crystalline  precipitate  with  iodin  in 
hydriodic  acid,  a  red  coloration  with  hydriodic  acid,  and  a  color  test 
similar  to  that  of  strychnin  with  sulphuric  acid  and  potassium  bi- 
chromate. This  substance  was  strongly  poisonous,  but  did  not  pro- 
duce the  tetanic  convulsions  characteristic  of  strychnin.  Ciotta  pro- 
nounced this  substance  as  probably  identical  with  strychnin,  but 
Selmi,  to  whom  portions  of  the  body  were  subsequently  submitted, 
found  that  the  substance  giving  the  color  reaction  was  not  crystalline, 
and  that  there  was  only  the  presumption  of  a  bitter  taste  to  it,  while 


240  IMPORTANCE  TO  TOXICOLOQIST. 

one  part  of  strychnin  in  forty  thousand  parts  of  water  is  intensely 
bitter.  Selmi  also  held  that  many  ptomains  give  reactions  similar  to 
strychnin  with  iodin  in  hydriodic  acid  and  with  hydriodic  acid  alone. 
He  also  held  that  the  physiological  properties  of  this  substance  were 
such  that  it  could  not  be  strychnin.  It  could  hardly  have  been  as- 
pidospermin,  which  reacts  with  sulphuric  acid  and  potassium  bichro- 
mate similarly  to  strychnin,  because  quebracho  bark,  in  which  this  alka- 
loid is  found  was  not  at  that  time  used  as  a  medicine  or  known  in  Italy. 

Ptomains  giving  reactions  similar  to  those  of  strychnin,  and  also 
causing  tetanic  spasms,  have  been  found  in  decomposed  corn-meal, 
and  Selmi  obtained  one  of  these  substances  which  differed  from 
strychnin  inasmuch  as  it  could  not  be  extracted  with  ether. 

Lombroso  named  the  poisonous  substance  found  in  decomposed 
corn-meal  "  pellagrocein,"  but  this  is  a  mixture  of  ptomains,  some  of 
which  produce  narcosis  and  paralysis,  and  others  induce  the  symp- 
toms of  nicotin  poisoning  instead  of  the  spasms  caused  by  strychnin. 

Morphin-like  Substances. — In  the  Sonzogna  trial  at  Cremona, 
Italy,  the  experts  seem  to  have  confounded  a  ptomain  with  morphin. 
This  substance  was  removed  from  either  alkaline  or  acid  solutions 
with  ether,  but  could  be  extracted  with  amylic  alcohol.  It  reduced 
iodic  acid,  but  in  its  other  reactions,  also  in  its  physiological  proper- 
ties, it  bore  no  resemblance  to  morphin.  In  frogs  it  arrested  the 
heart  in  systole,  which  is  said  never  to  happen  in  poisoning  with 
morphin.  It  failed  to  give  both  the  ferric  chlorid  and  the  Pellagri 
test  for  morphin. 

In  the  same  body  there  was  found  a  substance  which  was  ex- 
tracted from  alkaline  solutions  with  ether,  and  which  gave,  with 
hydrochloric  acid  and  a  few  drops  of  sulphuric  acid,  on  the  applica- 
tion of  heat,  a  reddish  residue  similar  to  that  obtained  by  the  same 
reagents  with  codein,  but  in  its  other  reactions  it  did  not  resemble 
this  alkaloid. 

Many  of  the  tests  for  morphin  employed  by  toxicologists  are  fal- 
lacious. In  the  examination  of  a  stomach  and  part  of  a  liver,  sent 
from  Lincoln,  Neb.,  Vaughan,  following  the  method  of  Dragendorff, 
obtained  in  the  amylic  alcohol  extract  from  alkaline  solution  a  resi- 
due that  gave  with  more  or  less  distinctness  all  the  principal  color 
tests  for  morphin  ;  but  failing  to  obtain  crystals  that  could  be  identi- 
fied as  those  of  this  alkaloid,  the  absence  of  morphin  was  reported. 
Haines,  working  with  the  same  material,  obtained  similar  reactions ; 
he  also  was  unable  to  secure  the  crystals  and  made  a  negative  report. 
Afterward,  it  was  quite  positively  shown  that  death  had  been  caused 
in  this  case  by  a  blow  on  the  back  of  the  head  and  that  no  morphin 
or  other  drug  had  been  administered. 

In  the  Buchanan  case  in  New  York,  the  symptoms  as  sworn  to  by 
the  attending  physician  clearly  were  not  those  of  morphin,  and  all 


MOBPHIN-LIKE  SUBSTANCES.  241 

the  tests  obtained  by  the  experts  were  duplicated  with  putrefactive 
products.  Many  skillful  chemists  have  carried  companion  portions 
of  decomposed  tissue,  one  portion  with,  and  the  other  without  mor- 
phin,  through  the  process  of  extraction  recommended  by  Dragendorflf, 
and  have  obtained  satisfactory  results,  finding  that  the  proper  residue 
responds  to  the  color  test  in  the  one  instance  and  fails  to  do  so  in 
the  other.  Tissues  have  been  thus  tested  in  apparently  every  stage 
of  putrefaction,  and  the  results  have  been  satisfactory  and.  confirma- 
tory of  the  methods  generally  employed.  There  is  one  point,  how- 
ever, which  has  been  constantly  overlooked.  The  putrefaction  to 
which  the  tissues  in  these  experiments  are  subjected  has  been  aero- 
bic, while  that  occurring  in  the  dead  body  is  anaerobic  ;  consequently, 
the  putrefactive  products  are  not  the  same  in  the  two  cases.  In  all 
experimental  studies  of  the  value  of  the  tests  for  morphin  in  decom- 
posing tissue,  putrefaction  must  be  allowed  to  proceed  in  the  absence 
of  oxygen.  There  is  a  second  point,  probably  of  equal  importance, 
and  this  concerns  the  kind  of  tissue  employed.  The  upper  portion 
of  the  small  intestines  (and  the  adjacent  tissue  after  death)  has  a 
bacterial  flora  peculiar  to  itself.  These  tissues  are  the  ones  quite 
universally  examined  in  medico-legal  cases,  and  consist  of  the  small 
intestine  itself,  the  stomach,  the  liver,  the  pancreas,  the  spleen,  and, 
in  some  instances,  the  kidneys.  Of  course,  the  bacteria  present  in 
the  small  intestine  during  life  may  after  death  extend  into  the  ab- 
dominal and  thoracic  viscera. 

Vaughan  allowed  finely-chopped  ox-liver  to  ferment  for  thirty 
days  under  anaerobic  conditions  and  then  divided  the  decomposed 
mass  into  three  portions,  which  were  marked  A,  B  and  C  To  B,  130 
mg.  of  morphin  sulphate  was  added,  and  to  C  the  same  amount  of 
morphin  together  with  0.5  gram  each  of  indol,  skatol  and  phenol; 
no  addition  was  made  to  A.  These  portions  were  carried  simulta- 
neously through  the  manipulations  recommended  by  Dragendorflf. 
To  the  residues  obtained  from  the  amylic  alcohol  extract  of  alkaline 
solutions,  in  which  the  chemist  expects  to  find  morphin,  all  the 
known  color  tests  for  morphin  were  applied  and  all  the  residues  re- 
sponded to  these  tests  in  the  same  manner.  In  all,  nitric  acid  gave 
a  lemon-brown  color  ;  sulphuric  acid  showed  no  change ;  sulphuric 
with  nitric  acid  gave  a  lemon-yellow,  slowly  changing  to  a  pink ; 
ferric  chlorid  gave  a  dirty  green  ;  iodic  acid  was  promptly  reduced ; 
Frohde's  reagent  gave  a  blue  color  without  any  violet ;  and  sulphuric 
acid  and  cane  sugar  produced  a  brownish-red  color,  changing  to  a 
wine-red.  These  investigations  show  that  the  color  tests  for  mor- 
phin, employed  by  toxicologists  in  following  the  scheme  of  Dragen- 
dorff,  are  wholly  untrustworthy.  The  substances  in  these  extracts 
which  vitiate  the  tests  for  morphin  probably  consist  of  indol  and  its 
derivatives.  Germs  which  produce  indol  and  kindred  substances 
are  native  and  constant  representatives  of  the  bacterial  flora  of  the 
16 


242  IMPORTANCE  TO  TOXICOLOGIST. 

upper  portion  of  the  small  intestine.  There  are  many  indol-forming 
bacteria,  and  while  some  of  these  may  be  present  in  any  tissue,  they 
are  certainly  present  in  health  and  in  disease,  during  life  and  after 
death,  in  the  small  intestine. 

Indol  and  its  derivatives  are  products  of  anaerobic  putrefaction  and 
this  accounts  for  the  fact  that  the  reactions  obtained  in  the  above- 
mentioned  experiments  are  not  familiar  to  those  toxicologists  who 
have  employed  tissue  allowed  to  putrefy  in  the  presence  of  oxygen. 
It  should  be  remembered  that  indol  may  be  prepared  synthetically 
and  it  may  be  obtained  from  putrefying  tissue.  Samples  obtained 
from  the  two  sources  do  not  always  give  exactly  the  same  reactions. 
Skatol  is  another  product  of  anaerobic  putrefaction  which  may  inter- 
fere with  the  tests  for  morphin.  While  it  would  be  comparatively 
easy  to  distinguish  pure  morphin  from  either  indol  or  skatol,  the 
results  of  the  above-mentioned  experiments  show  that  the  separation 
of  morphin  from  tissue,  decomposing  in  the  absence  of  oxygen,  and 
its  identification  are,  by  the  methods  now  generally  employed,  so 
uncertain  that  the  conscientious  chemist  will  seek  for  methods  free 
from  these  sources  of  error  before  he  gives  positive  testimony  of  the 
presence  of  this  alkaloid.  We  have  spoken  of  indol  and  its  deriva- 
tives as  being  present  in  decomposing  tissue,  and  it  should  be  stated 
that  the  nmnber  of  known  indol  derivatives  is  by  no  means  small, 
and  how  many  others  there  may  be  which  remain  unknown  no  one 
can  tell.  Many  of  these  substances  give  brilliant  color  reactions. 
Indoxyl  is  an  easily  decomposable  substance,  which  gives  some 
striking  color  reactions,  among  which  may  be  mentioned  the  produc- 
tion of  indigo  blue  with  ferric  chlorid  in  the  presence  of  free  hydro- 
chloric acid. 

Skatol-carbonic  acid  is  another  product  of  putrefaction  and  E.  and 
H.  Salkowski  obtained  1.3  grams  from  2  kg.  of  moist  fibrin  after 
twenty-six  days'  putrefaction.  Among  the  known  color  reactions  of 
this  substance  Hoppe-Seyler  mentions  the  following  : 

1.  If  a  dilute  solution  of  this  acid  (1:1000)  be  treated  with  a  few 
drops  of  pure  hydrochloric  acid  of  1.2  specific  gravity,  and  then  with 
a  few  drops  of  potassium  nitrate  solution  (2  per  cent.),  a  cherry-red 
coloration  is  produced,  and  later  a  red  precipitate  falls. 

2.  If  such  a  solution  be  mixed  with  an  equal  volume  of  hydro- 
chloric acid,  and  then  a  few  drops  of  chlorid  of  lime  solution  (J  per 
cent.)  be  added,  a  purple-red  color  is  produced. 

3.  Treated  with  a  few  drops  of  hydrochloric  acid,  then  with  two 
or  three  drops  of  a  very  dilute  solution  of  ferric  chlorid  and  heated, 
the  mixture  becomes  intensely  violet  before  boiling.  Skatol-carbonic 
acid  is  non-volatile. 

Skatol-acetic  acid  has  been  obtained  by  Nencki  by  the  anaerobic 
putrefaction  of  serum  albumin.  The  aqueous  solutions  of  this  sub- 
stance give  with  ferric  chlorid  a  white  cloudiness,  which  on  warm- 


MOBPHIN-LIKE  SUBSTANCES.  243 

ing  becomes  a  brick-red,  and  in  more  concentrated  solution  fire  red. 
Both  indigo  red  and  indigo  blue  may  be  formed  by  oxidation  of  indol. 

Knowing  that  indol  and  its  derivatives  are  formed  in  anaerobic 
putrefaction,  and  that  in  Dragendorff's  scheme  for  the  separation 
and  identification  of  vegetable  alkaloids,  these  substances  appear  in 
the  residues  which  are  tested  for  morphin,  and  knowing  the  great 
number  and  variety  of  color  reactions  given  by  these  substances,  it 
may  be  asked.  How  much  reliance  can  be  placed  on  the  color  tests 
for  morphin  ?  Besides  the  indol  bodies,  other  substances  are  formed 
in  the  anaerobic  putrefaction  of  proteid  bodies  and  among  these  are 
certain  aromatic  products  of  the  putrefaction  of  tyrosin.  The  fol- 
lo^ving  may  be  mentioned  :  (1)  Hydroparacumaric  acid  (paraoxy- 
phenylpropionic  acid).  This  substance  gives  with  ferric  chlorid  a 
distinct  but  evanescent  blue  coloration.  (2)  Paraoxyphenylacetic 
acid.  This  substance  gives  with  ferric  chlorid  a  pale  grayish-violet, 
which  soon  changes  to  a  dirty  green  color. 

Among  other  products  of  anaerobic  putrefaction  of  proteids,  phenol 
and  parakresol  may  be  mentioned  ;  with  the  former  ferric  chlorid 
gives  a  violet  color  and  with  the  latter  a  blue. 

In  the  case  of  Dr.  Urbino  de  Freitas  ^  of  Oporto,  Portugal,  who 
was  accused  of  attempting  the  wholesale  murder  of  his  wife's  family 
by  the  administration  of  morphin,  the  toxicologists  relied  for  the  de- 
tection of  this  alkaloid  upon  the  color  reactions  applied  to  residues 
obtained  by  following  Dragendorff's  method.  They  also  reported 
the  presence  of  narcein  and  the  probable  presence  of  delphinin. 
The  defense  questioned  the  reliability  of  the  tests  used  and  a  large 
number  of  the  most  prominent  toxicologists  in  Europe  became  in- 
volved in  the  controversy.  It  was  claimed  by  the  prosecution  that 
200  mg.  of  morphin  had  been  recovered.  On  this  point  Beckurts 
stated  that  it  was  incomprehensible  to  him  that  a  toxicologist  should 
recover  200  mg.  of  morphin  and  not  be  able  to  present  a  part  of  it 
in  evidence,  and  also  to  furnish  a  portion  to  the  experts  on  the  other 
side.  Brieger  and  Bischoff  thought  200  mg.  a  fabulous  quantity  to 
recover  and  that  a  portion  of  the  poison  should  have  been  presented 
in  evidence.  Husemann  thought  the  tests  relied  upon  insufficient, 
and  that  putrefactive  substances  had  been  mistaken  for  the  vegetable 
alkaloid,  and  even  Dragendorff  himself  thought  it  likely  that  this 
mistake  had  been  made.  For  the  detection  of  the  morphin  the  toxi- 
cologists for  the  prosecution  had  relied  chiefly  upon  the  application 
to  the  amylic  alcohol  residue  of  the  tests  with  iodic  acid,  Frohde's 
reagent,  and  sulphoselenic  acid  (known  as  Lafon's  reagent).  The 
chemists  consulted  quite  generally  agreed  that  these  tests  are  unreli- 
able and  that  the  amylic  alcohol  extract  of  putrefying  material  fre- 
quently responds  to  these  tests  when  morphin  is  known  not  to  be 
present. 

'  Relation  medico-legak  de  L' affaire  Urbino  de  Freitas. 


244  IMPORTANCE  TO  TOXICOLOGIST. 

Atropin-like  Substances. — Many  investigators  have  found  putre- 
factive products  which  in  their  mydriatic  properties  resemble  atropin 
and  hyoscyamin.  To  this  class  belongs  the  substance  observed  by 
Zuelzer  and  Sonnenschein.  It  was  removed  from  alkaline  solutions 
by  ether,  and  formed  microscopic  crystals,  an  aqueous  solution  of 
which,  when  applied  to  the  conjunctiva,  produced  a  mydriatic  effect, 
and,  when  administered  internally,  increased  the  action  of  the  heart 
and  arrested  the  movements  of  the  intestines.  Moreover,  in  its  beha- 
vior with  certain  alkaloidal  reagents,  such  as  platinum  chlorid,  it  re- 
sembled atropin ;  but  when  heated  with  sulphuric  acid  and  oxidizing 
agents  it  did  not  give  the  odor  of  blossoms  (Reuss's  test).  How- 
ever, Selmi  found  ptomatropins  which  with  sulphuric  acid  and  oxi- 
dizing agents  did  give  the  blossom  odor  as  distinctly  as  the  vegetable 
atropin.  These  putrefactive  bases  also  developed  this  odor  sponta- 
neously after  standing  for  two  or  three  days,  and  this  does  not 
happen  with  atropin.  The  odor  was  produced  with  the  ptomatro- 
pins by  nitric  and  sulphuric  acids  both  in  the  cold  and  on  the  appli- 
cation of  heat,  while  these  acids  in  the  cold  did  not  produce  the  odor 
with  atropin.  Ptomatropins  have  been  found  in  decomposing  fish, 
corned  beef,  putrid  game,  and  poisonous  sausage.  It  is  not  known 
whether  there  is  only  one  or  more  of  these  poisons.  The  symptoms 
often  resemble  those  of  belladonna  poisoning  very  closely.  The 
throat  becomes  dry,  the  muscles  of  deglutition  seem  to  be  paralyzed, 
the  secretion  of  perspiration  and  saliva  is  arrested,  mydriasis  may 
be  marked,  and  there  may  be  paralysis  of  accommodation,  ptosis,  and 
strabismus.  In  some  instances  delirium  and  in  others  convulsions 
appear.  The  heart-beat  becomes  rapid  and  weak.  The  tongue  is 
coated,  and  in  the  most  dangerous  cases  constipation  is  obstinate. 
The  general  weakness  may  be  extreme,  and  the  voice  wholly  lost. 
Section  shows  the  pharynx  swollen,  hemorrhagic  spots  in  the  oesoph- 
agus, stomach  and  intestines,  cloudy  swelling  of  the  solitary  fol- 
licles and  Peyer's  patches,  and  degeneration  of  the  heart  muscle. 
The  brain,  lungs,  and  kidneys  are  often  hyperemic.  Extracts  of 
putrid  material  will  often  cause  more  or  less  dilatation  of  the  pupil 
in  the  lower  animals  when  applied  locally.  Haines  and  Vaughan 
were  once  appointed  a  commission  to  inquire  into  the  tests  obtained 
by  a  toxicologist  who  had  reported  four  grains  of  atropin  in  the 
stomach  of  a  man  who  had  been  dead  for  some  weeks.  The  chief 
test  relied  upon  by  the  chemist  was  that  an  ounce  of  extract  from 
the  stomach  dilated  a  cat's  pupil  about  as  much  as  a  solution  of  four 
grains  per  ounce  of  atropin  sulphate.  It  is  needless  to  comment  on 
the  validity  of  such  evidence. 

Digitalin-like  Substances. — R5rsch  and  Fasbender  discovered  a 
putrefactive  base  which  affected  animals  very  much  like  digitalin. 
Trottarelli  obtained  a  similar  substance  from  the  brain  of  a  man  in 


A  DELPHININ-LIKE  SUBSTANCE.  245 

whose  abdominal  viscera  he  could  find  no  poison.  The  sulphate  of 
this  base  gave  on  evaporation  an  aromatic  smelling  and  astringent 
tasting  residue.  It  became  purple  with  sulphuric  acid  only,  and 
dark  red  with  hydrochloric  and  sulphuric  acids.  On  frogs  this 
ptomain  had  no  toxic  action. 

A  Veratrin-like  Substance. — Brouardel  and  Boutmy  obtained 
from  a  corpse  which  had  lain  in  water  for  eighteen  months,  and  a  large 
portion  of  which  had  changed  into  adipocere,  a  ptomain  resembling 
veratrin.  It  was  removed  from  alkaline  solution  by  ether,  and  on 
being  heated  with  sulphuric  acid  it  became  violet.  With  a  mixture 
of  sulphuric  acid  and  barium  peroxid  it  became,  in  the  cold,  brick- 
red  ;  and,  on  being  heated,  violet.  With  boiling  hydrochloric  acid 
it  took  on  a  cherry-red  coloration.  However,  it  differed  from  vera- 
trin, inasmuch  as  it  reduced  ferric  salts  instantly,  and  when  injected 
into  frogs  subcutaneously  it  did  not  induce  the  spasmodic  muscular 
contractions  characteristic  of  veratrin.  Bechamp  obtained,  by  the 
Stas-Otto  method  from  the  products  of  the  digestion  of  fibrin,  an 
alkaloidal  body  which  gave  with  sulphuric  acid  a  beautiful  carmine- 
red,  similar  to  that  given  with  gastric  juice,  and  again  extracting,  he 
obtained  a  body  which  behaved  with  sulphuric  acid  similarly  to 
curarin. 

A  Delphinin-like  Substance. — In  1870  Gibbone,  an  Italian  of 
prominence,  died  suddenly,  and  his  servant  was  accused  of  having 
poisoned  him.  Two  chemists  reported  the  presence  of  delphinin 
in  the  viscera,  but  it  seemed  improbable  that  the  servant  should 
know  anything  of  so  rare  a  substance,  or  that  he  should  have  been 
able  to  obtain  it.  However,  two  or  more  varieties  of  staphisagria 
grow  in  southern  Italy,  and  it  was  possible  that  the  servant  had 
used  some  preparation  made  by  himself  from  the  plant.  The  sup- 
posed alkaloid  was  given  to  Selmi  for  further  study.  It  was 
removed  from  alkaline  solutions  with  ether.  When  heated  with 
phosphoric  acid  it  became  red,  and  when  brought  into  contact  with 
concentrated  sulphuric  acid  reddish-brown.  In  these  tests  the  sub- 
stance resembled  delphinin,  but  with  sulphuric  acid  and  bromin 
water,  the  colorations  characteristic  of  the  vegetable  poison  failed  to 
appear.  Moreover,  Selmi  showed  that  delphinin  gives  the  following 
reactions  to  which  the  suspected  substance  does  not  respond  : 

1 .  Delphinin  dissolved  in  ether,  and  treated  with  a  freshly  pre- 
pared ethereal  solution  of  platinic  chlorid,  gives  a  white  flocculent 
precipitate,  which  is  insoluble  in  an  equal  volume  of  absolute  alcohol. 

2.  Delphinin  gives  precipitates  with  auro-sodium  hyposulphite, 
and  with  a  sulphuric  acid  solution  of  cupro-sodium-hyposulphite, 
the  latter  precipitate  being  soluble  in  an  excess  of  the  reagent. 
Finally  Ciaccia  and  Vella  showed  that  while  delphinin  arrests  the 


246  IMPORTANCE  TO  TOXICOLOOIST. 

heart  of  the  frog  in  diastole,  the  suspected  substance  arrests  it  in 
systole. 

A  Colchicin-like  Substance. — Baumert  found,  in  a  case  of  sus- 
pected poisoning,  twenty-two  months  after  death,  a  substance  giving 
many  of  the  reactions  for  colchicin.  It  was  extracted  from  acid 
solutions  with  ether,  to  which  it  imparted  a  yellow  color.  On 
evaporation  of  the  ether,  a  yellow,  amorphous  substance  remained, 
and  this  dissolved  in  warm  water  with  yellow  coloration.  It  could 
be  extracted  from  acid  solutions  also  by  chloroform,  benzol  and 
amylic  alcohol,  but  not  from  petroleum  ether.  It  was  removed  with 
much  more  difficulty  from  alkaline  solutions.  All  the  extracts  were 
yellow  and  left  on  evaporation  a  strictly  alkaline,  markedly  bitter, 
sharp  tasting,  amorphous  yellow  residue,  which  dissolved  in  water 
and  dilute  acids  incompletely,  forming  a  resin.  When  this  resin 
was  dissolved  in  dilute  sodium  hydrate  and  the  solution  rendered 
acid  with  sulphuric  acid,  the  same  reactions  were  obtained  as  with 
the  original  extract.  With  phosphomolybdic  acid,  phosphotungstic 
acid,  potassio-bismuthic  iodid,  potassio-mercuric  iodid,  iodin  in 
potassium  iodid,  cyanic  acid,  and  gold  chlorid,  this  substance  gave 
the  same  reactions  which  were  obtained  by  parallel  experiments  with 
colchicin.  Thus,  the  tannic  acid  precipitates  were  both  soluble  in 
alcohol,  and  the  precipitates  with  phosphomolybdic  acid  in  both 
cases  became  blue  on  the  addition  of  ammonium  hydrate.  Concen- 
trated sulphuric  and  dilute  nitric  and  hydrochloric  acids  dissolved 
the  supposed  colchicin  with  yellow  coloration.  Strong  nitric  acid 
colored  the  substance  dirty  red,  scarcely  to  be  called  a  violet.  When 
the  substance  was  purified  as  much  as  possible,  this  color  became  a 
beautiful  carmine-red.  The  addition  of  water  changed  the  red  into 
yellow,  and  caustic  soda  produced  a  dark  dirty  orange.  In  general, 
in  the  above-mentioned  reactions,  the  putrefactive  product  agreed 
with  colchicum,  but  the  former  gave  precipitates  with  picric  acid 
and  platinum  chlorid,  while  the  latter  gave  no  precipitates  with  these 
reagents. 

In  1886,  Zeisel  proposed  the  following  test  for  colchicum  :  When 
a  hydrochloric  acid  solution  of  the  alkaloid  is  boiled  with  ferric 
chlorid,  it  becomes  green,  sometimes  dark  green,  and  cloudy ;  then, 
if  the  fluid  be  agitated  with  chloroform,  the  chloroform  takes  up  the 
coloring  matter  and  appears  brownish-red,  or  dark,  and  the  super- 
natant fluid  clears  up  without  becoming  wholly  colorless.  Baumert 
applied  this  test  to  both  colchicum  and  the  putrefactive  product. 
To  from  2  to  5  c.c.  of  the  suspected  solution  in  a  test-tube,  he  added 
from  five  to  ten  drops  of  strong  hydrochloric  acid,  and  from  four  to 
six  drops  of  a  10  per  cent,  solution  of  ferric  chlorid,  then  heated  the 
mixture  directly  over  a  small  flame  until  it  was  evaporated  to  half 
its  volume  or  less.     In  the  presence  of  1  mg.  of  colchicin  the  origi- 


A   COLCIIICIN-LIKE  SUBSTANCE.  247 

nally  bright  yellow  solution  became  gradually  green,  and,  on  further 
concentration,  dark  green  and  cloudy ;  then,  on  shaking  the  fluid 
with  chloroform,  admitting  as  much  air  as  possible,  the  chloroform 
subsided,  having  a  red  color  if  as  much  as  2  mg.  of  colchicin  were 
present,  and  a  bright  yellow  color  if  only  1  mg.,  and  the  supernatant 
fluid  became  a  beautiful  olive  green.  When  either  petroleum  ether, 
benzol,  carbon  bisulphid,  or  amylic  alcohol  was  substituted  for  the 
chloroform  the  coloration  did  not  appear.  From  this  Baumert  in- 
fers that  the  red  coloring  matter  is  either  soluble  in  chloroform  or 
that  it  is  not  formed  until  the  chloroform  is  added.  He  found  that 
the  putrefactive  product  did  not  respond  to  this  test.  Some  of  this 
substance  was  sent  to  Brieger,  who  decided  that  it  was  not  a  base, 
but  a  pepton-like  substance ;  it  was  also  found  to  be  inert  physio- 
logically. 

Liebermann  found  a  similar  colchicin-like  substance  in  a  cadaver. 
His  description  differed  from  that  of  Baumert  only  in  regard  to  the 
taste  of  the  substance,  Liebermann  having  failed  to  observe  any 
marked  taste  in  his  body,  while  Baumert  reported  a  distinctly  bitter 
taste.  A  colchicin-like  substance  has  been  found  in  beer,  and  it  has 
been  suggested  that  it  was  this  that  the  above-mentioned  toxicolo- 
gists  found  in  the  bodies  which  they  examined,  but  Liebermann  states 
that  the  man  whose  body  he  analyzed  had  been  a  total  abstainer  from 
beer. 


CHAPTER    XIV. 

CHEMISTRY  OF   THE  PTOMAINS. 

The  basic  substances  described  in  the  following  pages  are  ar- 
ranged, as  far  as  possible,  in  the  regular  natural  order.  An  inspec- 
tion of  the  list  of  these  bases  will  show  the  remarkable  fact  of  the 
predominancy  of  the  amine  type.  Almost  two-thirds  of  the  known 
ptomains  contain  only  C,  H,  and  N,  and  represent  simple  ammonia 
substitution  compounds.  Of  the  oxygenated  bases,  all  of  those 
whose  constitution  is  known  possess  the  trimethylamin  group  as 
their  basic  constituent,  and  it  is  quite  probable  that  most,  if  not  all, 
of  the  remaining  ptomams  will  be  found  to  possess  the  same  or  a 
similar  basic  nucleus. 

It  will  be  seen,  furthermore,  that  a  very  large  number  of  the 
ptomains  described  possess  little  or  no  toxic  action,  and  are,  therefore, 
physiologically  inert.  It  would  seem,  as  Brieger  has  already  pointed 
out,  that  a  certain  quantity  of  oxygen  is  necessary  to  the  formation 
of  poisonous  bases.  A  free  supply  of  oxygen,  on  the  other  hand,  in- 
variably yields  non-toxic  ptomains.  The  poisonous  bases  begin  to 
appear  about  the  seventh  day  of  putrefaction,  and  in  turn  disappear 
if  this  is  allowed  to  go  on  for  a  considerable  period  of  time. 

It  has  already  been  pointed  out  that  the  ptomains  are  to  be  regarded 
as  extra-cellular  products  of  bacterial  activity.  They  do  not  origi- 
nate within  the  bacterial  cell  and  therefore  they  are  not  to  be  looked 
upon  as  direct  metabolic  products  of  the  cell  protoplasm  but  rather 
as  secondary  cleavage  products.  The  production  of  amines  and  di- 
amines, for  example,  is  clearly  due,  as  in  the  case  of  monamino  acids 
leucin  and  tyrosin,  to  the  splitting  off  of  preexisting  groups  in  the 
substance  acted  upon.  This  fact  has  been  made  especially  clear  in 
the  demonstration  of  the  origin  of  putrescin  and  cadaverin  from  the 
proteid  bases  arginin  and  lysin ;  and  of  cholin  and  allied  bases  from 
lecithin. 

Methylamin,  CHj.NHg;  this  is  the  simplest  organic  base  that 
is  formed  in  the  process  of  putrefaction.  It  is  ammonia  in  which 
one  atom  of  hydrogen  has  been  replaced  by  the  methyl  radical.  It 
occurs  in  herring-brine  (Tollens,  1866  ;  Bocklisch,  1885) ;  in  decom- 
posing herring,  twelve  days  in  spring  (Bocklisch) ;  in  pike,  six  days 
in  summer  (Bocklisch) ;  in  haddock,  two  months  at  a  low  tempera- 
ture (Bocklisch) ;  in  the  fermentation  of  cholin  chlorid  (Hasebroek). 
Brieger  found  it  present  in  cultures  of  comma  bacillus  on  beef-broth 

248 


DIMETHYLAMIK  249 

which  were  kept  for  six  weeks  at  37°-38°.  Ehrenberg  reported  its 
possible  presence  in  poisonous  sausage,  and  obtained  it  by  growing  a 
bacillus  from  this  source  on  intestines  (1887).  Morner  (1896)  found 
it  in  fermented  fish  associated  with  di-  and  tri-methylamins  and 
cholin.  Emmerling  (1 897)  found  it  with  the  latter  and  a  base  CgHj^N 
in  the  streptococcus  decomposition  of  fibrin.  In  Brieger's  method 
methylamin  is  found  in  both  the  mercuric  chlorid  precipitate  and  fil- 
trate. The  mercury  double  salt  is  readily  soluble  in  water,  and  can 
thus  be  separated  from  any  accompanying  cadaverin  or  putrescin. 
Methylamin  is  an  inflammable  gas  of  strong  ammoniacal  odor,  and 
burning  with  a  yellow  flame.  It  is  readily  soluble  in  water,  and  its 
solutions  give  reactions  similar  to  those  of  ammonia.  Its  salts  are, 
as  a  rule,  also  soluble  in  both  water  and  alcohol. 

The  hydrochlorid,  CII3.NII2.IICl,  crystallizes  in  large  deliques- 
cent plates.  On  being  heated  with  alkali,  it  gives  oif  the  odor  of 
methylamin. 

The  platinochlorid,  (CH3.NH2.HCl)2PtCl,  (Pt  =41.31  per  cent.),' 
yields  hexagonal  plates  which  usually  occur  heaped  up  in  several 
layers.  It  is  soluble  in  about  fifty  parts  of  water  at  ordinary  tem- 
perature, and  can  be  readily  recrystallized  from  hot  water.  It  is  in- 
soluble in  absolute  alcohol  and  in  ether. 

The  aurochlorid,  CHj.NH^.HCl.AuCls  -|-  HgO,  forms  prisms 
which  are  readily  soluble  in  water.  There  is  also  a  readily  soluble 
picrate. 

Methylamin  does  not  possess  any  toxic  action,  even  when  given 
in  fairly  large  doses.  This  physiological  indifference  is  shared  by 
nearly  all  the  monamins  and  diamins  that  have  been  obtained  among 
the  products  of  putrefaction. 

Dimethylamin,  (CH3)2.NII,  has  been  found  in  putrefying  gelatin, 
ten  days  at  35°  (Brieger,  1885) ;  in  yeast  decomposing  in  covered 
vessels  for  four  weeks  during  summer  (Brieger) ;  in  decomposing 
perch  six  days  in  summer  (Bocklisch) ;  and  in  herring-brine  (Bock- 
lisch,  1886);  in  fermented  fish  (Morner,  1896).  It  has  been  found 
in  poisonous  sausage,  and  in  cultures  of  a  bacillus,  obtained  from  this 
source,  on  liver  and  intestines  (Ehrenberg,  1887).  It  is  also  formed 
together  with  tri methylamin,  when  neuridin  hydrochlorid  is  distilled 
with  sodium  hydrate  (Brieger,  L,  23).  It  occurs  in  the  mercuric 
chlorid  precipitate  as  well  as  filtrate.  From  cadaverin  it  can  be  sep- 
arated by  platinum  chlorid,  since  cadaverin  platinochlorid  is  difficultly 
soluble  in  cold  water,  and  recrystallizes  from  hot  water,  whereas  the 
dimethylamin  double  salt  remains  in  the  mother-liquor.  In  like 
manner  it  can  be  separated  from  neuridin.     From  cholin  it  can  be 

^  The  percentages  given  in  the  following  pages  are  calculated  from  Au  =  196. 64 
(Kriiss),  Pt  =  19446  (Seubert),  CI  =  35.37,  0=15.96. 


250  CHEMISTRY  OF  THE  PTOMAINS. 

isolated  by  reciystallizing  the  mercuric  chlorid  precipitate  from  hot 
water. 

The  free  base  is  a  gas  at  ordinary  temperature,  but  can  be  con- 
densed to  a  liquid  which  boils  at  8°-9°.  The  hydrochlorid, 
(CH3)2.]SrH.HCl,  crystallizes  in  needles,  which  deliquesce  on  expo- 
sure to  air  and  are  soluble  in  absolute  alcohol  (Brieger,  L,  56).  It 
is  insoluble  in  absolute  alcohol  (Bocklisch),  but  soluble  in  chloro- 
form (Behrend),  and  can  then  be  separated  from  methylamin  hydro- 
chlorid, which  is  insoluble  in  chloroform. 

The  platinochlorid,  [(CH3)2.NH.HC1]  ^PtCl,  (Pt  =  39.00  per 
cent.),  crystallizes  in  long  needles,  which  are  easily  soluble  in  hot 
water,  less  soluble  in  cold  water.  Sometimes  it  forms  orange-yellow 
plates  or  prisms,  or  else  small  needles. 

The  aurochlorid,  (CH3)2.NH.HCl.AuCl3,  forms  needles  (Bock- 
lisch),  or  large  yellow  monoclinic  plates  (Hjortdahl),  which  are  in- 
soluble in  absolute  alcohol. 

With  mercuric  chlorid  it  unites  to  form  two  double  salts  (Morner). 

Trimethylamin,  CJil^  =  (CHg)^]^,  has  been  known  for  a  long 
time  to  occur  in  animal  and  vegetable  tissues.  Dessaignes  showed 
its  presence  in  leaves  of  Chenopodium  (1851),  in  the  blood  of  calves 
(1857),  and  later  in  human  urine.  It  has  been  obtained  from  ergot 
(Secale  cornutum)  by  Walz  (1852)  and  Brieger  (1886)  ;  from  herring- 
brine  by  Wertheim,  Winkles,  Tollens  and  Bocklisch.  In  these 
substances,  with  the  exception  of  herring-brine,  it  probably  does  not 
exist  pre-formed,  but  is  rather  a  product  of  the  method  employed  for 
its  isolation.  In  fact,  Brieger  has  shown  that  it  does  not  exist  in 
ergot,  but  is  formed  at  the  expense  of  the  cholin  present,  which,  on 
distillation  with  potash,  decomposes  and  yields  trimethylamin  and 
glycol.     Thus : 

C2H,OH.N(CH3)3.0H  =  N(CH3)3  +  Q^B.^{OK)^. 

It  is  also  formed  when  betain  and  neuridin  are  distilled  with  potash. 
It  may  have  a  similar  origin  in  most  of  the  other  cases,  since  cholin 
is  now  known  to  be  widely  disseminated  in  plants  and  animals, 
either  as  such  or  as  a  constituent  of  the  more  complex  lecithin. 

Trimethylamin  has  been  found  in  the  putrefaction  of  yeast 
(Hesse,  1857  ;  Miiller,  1858);  in  cheese  after  six  weeks  in  mid- 
summer (Brieger);  in  human  liver  and  spleen  after  from  two  to  seven 
days  (Brieger);  in  perch  after  six  days  in  midsummer  (Bocklisch) ; 
in  mussel  (Mytilus  edulis)  after  sixteen  days  (Brieger);  in  putrefying 
brains  after  from  one  to  two  months,  and  in  fresh  brains  (Guareschi 
and  Mosso);  in  cultures  of  the  streptococcus  pyogenes  on  beef-broth, 
bouillon,  meat-extract,  and  blood-serum ;  from  cultures  of  the 
comma-bacillus  (Brieger),  and  from  cultures  of  proteus  vulgaris  (Car- 
bone).  It  has  also  been  found  in  cod-liver  oil.  Ehrenberg  (1887) 
reports  its  presence  in  considerable  quantity  in  poisonous  sausage, 


ETHYLAMIN.  251 

and  in  cultures  of  a  bacillus,  isolated  from  this,  grown  on  liver, 
intestines,  and  meat-bouillon.  Stadthagen  has  found  it  in  normal 
urine ;  Kulueff  in  the  feces  of  a  case  of  gastroptosis.  Morner  ob- 
tained it  together  with  cholin  and  the  preceding  amins  from  "  fer- 
mented fish,"  an  article  of  food  consumed  in  northern  Sweden 
(1896).  Emmerling  found  it  in  putrefying  gluten,  also  in  strepto- 
coccus decomposition  of  fibrin  together  with  methylamin  and  the 
base  CgH^jN  (1897). 

Trimethylamin  is  found  in  both  the  mercuric  chlorid  precipitate 
and  filtrate.  It  remains  in  the  mother-liquor  from  which  cadaverin, 
neuridin,  and  dimethylamin  platinochlorids  have  crystallized.  If 
an  aqueous  solution  of  mercuric  chlorid  is  used  as  the  precipitant, 
the  trimethylamin  will  be  found  almost  entirely  in  the  filtrate,  from 
which  it  can  be  obtained  after  removal  of  the  mercury  by  evaporat- 
ing the  filtrate  to  dryness,  extracting  with  alcohol,  and  treating  the 
solution  thus  obtained  with  alcoholic  platinum  chlorid. 

The  free  base  is  a  liquid  possessing  a  strong,  fish-like  odor.  Its 
boiling-point  is  9.3°.  It  is  strongly  alkaline  in  reaction  and  freely 
soluble  in  water. 

The  hydrochlorid,  (CH3)3N.HC1,  is  deliquescent  and  freely  soluble 
in  water  and  alcohol.  Heated  to  285°  it  decomposes.  With  alkalis 
it  gives  off  the  odor  of  the  free  base. 

The  platinochlorid,  [(CH3)3N.HCl]2PtCl,(Pt  =  36.92  per  cent.), 
is  soluble  in  hot  water,  from  which,  on  cooling,  it  recrystallizes  in 
orange-red  octahedra  or  needles,  which  do  not  lose  water  when  heated 
at  100°-110°  (Bocklisch). 

The  aurochlorid,  (CH3)3N.HCl.AuCl3  (Au  =  49.39  per  cent.),  is 
easily  soluble,  and  hence  can  be  separated  from  cholin  aurochlorid, 
which  is  difficultly  soluble.  Similarly  this  base  can  be  separated 
from  ammonia  by  the  use  of  gold  chlorid.  The  mercurochlorid  con- 
tains two  molecules  of  HgClg  (Morner). 

Trimethylamin  is  not  a  strong  j)oison,  since  very  large  doses  of  it 
must  be  given  in  order  to  bring  out  physiological  disturbances. 

Ethylamin,  CgHg.NH,,  is  formed  in  putrefying  yeast  (Hesse, 
1857)  ;  in  wheat  flour  (Sullivan,  1858) ;  and  also  in  the  distillation 
of  beat-sugar  residues. 

It  is  a  strongly  ammoniacal  liquid  boiling  at  18.7°  and  is  miscible 
with  water  in  every  proportion.  Like  the  other  amins,  it  is-  com- 
bustible. It  possesses  strong  basic  properties,  and  is  capable  of  ex- 
pelling ammonia  from  its  salts  in  a  manner  analogous  to  the  action 
of  the  fixed  alkalis. 

The  hydrochlorid,  CgH^.NHg.HCl,  forms  deliquescent  plates, 
which  melt  at  76°-80°.     It  is  readily  soluble  in  water  and  alcohol. 

The  platinochlorid,  (C2Hg.]SrH2.HCl)2PtCl^,  forms  orange-yellow 
rhombohedra  ( Weltzien),  or  hexagonal-rhombohedral  crystals  (Topsoe). 


252  CHEMISTRY  OF  THE  PTOMAINS. 

The  aurochlorid,  CjHg.NHj.HCl.AuClg,  forms  gold-yellow  mono- 
clinic  prisms,  readily  soluble  in  water. 

AVith  picric  acid  it  forms  short  brown  prisms,  not  very  soluble  in 
water. 

Diethylamin,  C^Hj^N  =  (C2H5)2NH,  has  been  obtained  by  Bock- 
lisch  from  pike  which  were  allowed  to  putrefy  for  six  days  in  sum- 
mer ;  and  by  growing  a  bacillus  obtained  from  poisonous  sausages  on 
intestines  and  on  meat-bouillon  (Ehrenberg,  1887). 

It  is  an  inflammable  liquid  which  boils  at  57.5°,  possesses  strong 
basic  properties,  and  is  soluble  in  water. 

The  hydrochlorid,  (C2H5)2NH.HC1,  crystallizes  in  needles 
(Bocklisch)  ;  in  long  needles  and  prisms  from  absolute  alcohol ;  in 
plates  from  ether-alcohol.  These  are  not  deliquescent  and  are  easily 
soluble  in  water  and  in  chloroform  ;  rather  difficultly  in  absolute  alco- 
hol. Heated  with  sodium  hydrate  it  gives  off  alkaline  vapors.  From 
an  alcoholic  solution  it  is  precipitated  by  addition  of  alcoholic  mercuric 
chlorid.  The  mercury  double  salt  is  difficultly  soluble  in  hot  water, 
from  which  it  recrystallizes  on  cooling. 

The  platinochlorid,  [(C2H,)2.NH.HCl]2PtCl4,  crystallizes  in 
orange-yellow  monoclinic  crystals  which  are  easily  soluble  in  water. 

The  aurochlorid,  (C2H5)2NH.HCl.AuCl3 (Au  =  47.71  percent.), 
forms  tri metric  crystals  (Topsoe),  which  are  difficultly  soluble  (Bock- 
lisch).    It  melts  at  about  165°. 

With  picric  acid  it  forms  an  easily  soluble  picrate  (Lea). 

Triethylamin,  CgH^jN  =  (C2ll5)3N,  was  obtained  by  Brieger 
(1885)  from  haddock  which  were  exposed  for  five  days  in  an  open 
vessel  during  summer.  He  obtained  it  by  distilling  with  potash, 
after  removal  of  platinum  by  hydrogen  sulphid,  the  mother-liquor 
from  which  neuridin,  the  base  C2Hg]S[2,  muscarin,  and  gadinin  had 
successively  crystallized  (see  gadinin).  It  has  also  been  found  by 
Bocklisch  (1886)  in  putrid  pike,  and  by  Ehrenberg  (1887).  The 
latter  obtained  it  from  cultures  of  a  bacillus,  found  in  poisonous  sau- 
sage, and  grown  on  meat-bouillon. 

The  free  base  is  oily  in  character  and  possesses  an  ammoniacal 
odor.     It  is  but  slightly  soluble  in  water,  and  boils  at  89°- 89.5°. 

The  platinochlorid,  [(C2H,)3N.HCl]2PtCl,  (Pt  =  31.84  per 
cent.),  crystallizes  in  needles,  which  are  readily  soluble  in  water. 

With  mercuric  chlorid  the  aqueous  solution  gives  no  precipitate. 

With  picric  acid  it  yields  yellow  needles  which  are  but  slightly 
soluble  in  cold  water. 

Propylamin,  C^Hy.NHj,  is  isomeric  with  trimethylamin,  and  can 
therefore  be  easily  confounded  with  that  base.  There  are  two  pro- 
pylamins  possible  represented  by  the  formulae  CHj.CHj.CHj.NHj 


CAPBOYLAMIN.  253 

and  (CH3)2.CH.NH2.  The  former,  or  the  normal  compound,  boils 
at  47°-48°,  whilst  the  latter,  or  iso-propylamin,  boils  at  31.5°. 
Both  are  liquids  possessing  an  ammoniacal,  fish-like  odor.  They 
form  crystalline  salts ;  the  hydrochlorids  melt  respectively  at  155°- 
158°  and  at  139.5°. 

Iso-propylamin  (?)  has  been  found  among  the  distillation  products 
of  the  vinasse  of  beet-root  molasses.  Propylamin  has  been  obtained 
by  Brieger  (1887)  from  cultures  of  the  bacteria  of  human  feces  on 
gelatin.  Schwanert  has  isolated  from  the  organs  of  a  cadaver  a 
basic  substance  which  was  said  to  possess  an  odor  similar  to  propyl- 
amin. 

Butylamin,  C^H^jN,  was  obtained  by  Gautier  and  Mourgues 
(1888)  in  cod-liver  oil.  It  forms  a  colorless,  mobile,  alkaline  liquid, 
the  boiling  point  of  which  they  found  to  be  86°  at  760  mm.  It 
absorbs  carbonic  acid  from  the  air  and  readily  forms  salts.  The 
platinochlorid  forms  golden-yellow  plates  which  are  quite  soluble. 

In  animals  it  produces  an  increase  in  the  function  of  the  skin  and 
kidneys,  and  in  large  doses  fatigue,  stupor  and  vomiting. 

Iso-amylamin,  C.HjjN  =  (CH3)3.CH.CH2.CH2.NH2,  has  been 
obtained  by  Limpricht  in  the  distillation  of  horn  with  potash  ;  it 
also  occurs  in  the  putrefaction  of  yeast  (Miiller,  Hesse,  1857);  and 
in  cod-liver  oil  (Gautier  and  Mourgues,  1888),  where  it  constitutes 
nearly  one-third  of  the  bases  present.  Since  leucin  on  heating  yields 
amylamin  and  carbonic  acid  it  may  be  looked  upon  as  a  source  of 
this  ptomain. 

It  is  a  colorless,  strongly  alkaline  liquid,  possessing  an  odor  which 
is  not  disagreeable.  At  the  ordinary  pressure  it  boils  at  97°- 
98°. 

The  hydrochlorid  forms  deliquescent  crystals,  which  have  a  bitter, 
disagreeable  taste.  The  platinochlorid  crystallizes  in  golden-yellow 
slender  plates,  which  are  very  soluble  in  boiling  water.  The  base 
is,  according  to  Gautier  and  Mourgues,  identical  with  that  obtained 
by  treating  iso-amylcarbimid  with  potash. 

It  is  a  very  active  poison,  producing  rigor,  convulsions,  and  death. 
Four  milligrams  produce  death  in  a  greenfinch  in  three  minutes. 

Caproylamin  (Hexylamin),  CgHj^JST,  has  been  found  to  occur  by 
Hesse  (1857)  in  the  putrefaction  of  yeast.  Hager  isolated  from 
some  putrid  material  what  he  thought  to  be  a  mixture  of  amylamin 
and  caproylamin,  and  named  it  septicin. 

Hexylamin  was  found,  in  small  quantity,  in  cod-liver  oil,  by 
Gautier  and  Mourgues,  and  according  to  these  authors  it  resembles 
amylamin  in  its  action,  but  is  less  toxic. 


254  CHEMISTRY  OF  THE  PTOMAINS. 

Tetanotoxin,  C^Hj^N(?),  was  obtained  by  Brieger  (1886)  as  one 
of  the  products  of  the  growth  of  the  tetanus  bacillus  on  beef-broth 
or  on  brain-broth.  It  has  also  been  obtained  by  Kitasato  and  Weyl 
(1890)  from  pure  cultures  of  the  tetanus  bacillus,  kept  eight  days  at 
36°.  For  its  isolation  see  tetanin  and  Ber.  d.  d.  chem.  Ges.,  19, 
3120.  It  is  tetanizing  in  action,  produces  first  tremor,  then  paral- 
ysis and  violent  convulsions.  It  forms  an  easily  soluble  gold  double 
salt  which  melts  at  130°.  The  platinochlorid  is  difficultly  soluble, 
and  decomposes  at  240°.  The  hydrochlorid  is  crystalline,  and  is 
readily  soluble  in  alcohol  and  in  water.  It  melts  at  about  205°. 
From  warm  alcohol  it  crystallizes  in  flat,  pointed  plates. 

Spasmotoxin,  a  base  of  as  yet  unknown  composition,  produces 
in  animals  violent  clonic  and  tonic  convulsions.  It  was  ob- 
tained by  Brieger  (1887)  from  cultures  of  the  tetanus  germ  on  beef- 
broth. 

Another  toxin  was  obtained  by  Brieger  (1887),  in  cultures  of  the 
tetanus  microbe,  which  produced  complete  tetanus,  salivation  and  tear- 
secretion.  In  its  composition  it  is  probably  a  diamin.  The  platin- 
ochlorid forms  plates  which  begin  to  decompose  at  240°.  The 
hydrochlorid  is  very  deliquescent.  Gold  chlorid  and  picric  acid 
form  very  soluble  compounds.  Besides  these  three  bases  he  isolated 
another  toxic  substance,  tetanin,  and  a  base  (see  under  tetanin). 

Dihydrolutidin,  CyH^iN,  was  found  in  cod-liver  oil  by  Gautier 
and  Mourgues  (1888).  It  is  the  first  known  hydrolutidin.  It  is  a 
colorless,  somewhat  oily,  very  alkaline  and  caustic  liquid,  the  odor 
of  which  is  sharp,  but  somewhat  agreeable  when  dilute.  It  absorbs 
carbonic  acid  from  the  air,  darkens  and  thickens ;  is  feebly  soluble 
in  water,  and  boils  at  199°  at  760  mm.  pressure.  The  salts  are 
bitter  to  the  taste. 

The  hydrochlorid  crystallizes  in  a  confused  mass  of  needles  or  in 
plates.  The  nitrate  reduces  silver  nitrate — a  property  of  all  hydro- 
pyridin  bases  (Hoffmann).  The  sulphate  forms  fine  stellate  deli- 
quescent needles. 

The  platinochlorid  is  readily  precipitated  from  concentrated 
solutions  as  a  canary-yellow  precipitate.  From  warm  solutions  it 
crystallizes  in  lozenge-shaped  plates  which  are  often  imbricated.  On 
boiling  with  water  it  loses  hydrochloric  acid  and  forms  (CyIIjjNCl)2. 
PtClg,  which  possesses  a  lighter  color,  is  more  soluble  than  the 
normal  salt,  and  crystallizes  confusedly. 

The  aurochlorid  crystallizes  in  needles  Avhich  form  fan-  or  lozenge- 
shaped  masses.     It  is  scarcely  altered  even  in  hot  water. 

The  iodomethylate,  C^HjjN.CHgl,  is  obtained  by  mixing,  in  the 
cold,  the  base  and  methyl  iodid.  The  colorless  compound  thus  ob- 
tained is  soluble  in  water  and  in  alcohol,  and  possesses  a  disagree- 


COLLIDIN  ISOMER.  255 

able,  somewhat  nauseating  odor.  Treated  with  potash  it  yields  a 
colorless,  aromatic,  very  alkaline  oil. 

The  base  on  oxidation  with  boiling  potassium  permanganate  yields 
an  acid,  CyHyN02,  and  from  this  fact  the  discoverers  conclude  that 
the  base  is  a  dihydro-dimethylpiridin,  C.H^(CH3)2NH. 

It  is  moderately  poisonous.  In  small  doses  it  diminishes  the  gen- 
eral sensibility  ;  in  larger  doses  it  produces  trembling,  especially  of 
the  head ;  profound  depression  alternating  with  periods  of  extreme 
excitement ;  paralysis  of  the  posterior  limbs,  and  death. 

A  Base,  CgHj^N,  isomeric  but  not  identical  with  aldehyde  coUidin, 
was  obtained  by  Nencki  as  early  as  1876,  by  allowing  a  mixture  of 
200  grams  of  pancreas  and  600  grams  of  gelatin  in  ten  liters  of 
water  to  putrefy  for  five  days  at  40°.  The  method  used  by  Nencki 
for  its  isolation  is  as  follows  :  The  fluid  mass  was  distilled  with  sul- 
phuric acid,  to  drive  off  the  volatile  acids,  then  rendered  alkaline 
with  barium  hydrate,  and  again  distilled.  The  distillate  was  re- 
ceived in  dilute  hydrochloric  acid,  and  on  evaporation  gave  a  crys- 
talline residue  of  ammonium  chlorid,  and  of  a  salt  which  formed 
long  rhombic  plates.  The  latter  were  separated  from  the  ammonium 
salt  by  absolute  alcohol.  The  free  base  was  obtained  from  the  salt 
by  treating  it  with  sodium  hydrate,  and  extracting  the  solution  with 
ether. 

This  compound,  as  already  stated,  is  isomeric  with  coUidin,  and 
also  with  O.  de  Coninck's  base,  with  which  it  is  possibly  identical. 
The  latter,  however,  will  be  described  separately. 

On  decomposing  fibrin  by  means  of  streptococci  Emmerling  (1897) 
obtained  besides  leucin,  tyrosin  and  various  organic  acids,  methyl- 
amin,  tri-methylamin  and  a  base,  CgHj^N,  which  apparently  differed 
from  that  of  Nencki  and  that  of  Gautier  and  Etard  by  the  ready 
solubility  of  its  platinum  salt.  It  had  no  effect  on  guinea-pigs.  It 
formed  a  syrupy  liquid,  and  gave  with  gold  chlorid  a  yellow  precipitate 
which  rapidly  became  reduced.  Platinum  chlorid  gave  no  precipi- 
tate but  the  mixture  on  slow  evaporation  gave  yellowish-red  crystals 
of  the  platinochlorid.  Concentrated  solutions  gave  a  picrate  which 
melted  at  172°-175°.  Owing  to  the  slight  pyridin  odor  of  the  base 
the  latter  was  regarded  as  a  propyl  pyridin. 

The  free  base  is  oily  in  character,  and  possesses  a  peculiar,  not 
unpleasant  odor.  It  readily  absorbs  carbonic  acid  gas  from  the  air, 
forming  after  a  time  a  lamellar,  crystalline  mass  of  the  carbonate. 
The  salt  of  this  base  on  heating  gives  off  an  oil  which  burns  with  a 
smoky  flame,  and  possesses  an  odor  similar  to  that  of  xylol  or  cumol. 
Nencki  was  therefore  at  first  of  the  opinion  that  the  ptomain  was  an 
aromatic  base,  probably  an  isophenyl-ethylamin  of  the    following 

<OIT 
^TT  •    He  supposed  that  it  was  formed 


256  CHEMISTRY  OF  THE  PTOMAINS. 

by  the  putrefaction  of  tyrosin,  according  to  the  following  equation  : 

C9H11NO3  =  CgHiiN  +  CO,  +  O. 

We  know  that  tyrosin  does  split  up,  on  being  heated  to  270°,  into 
carbonic  acid  and  oxyphenyl-ethylamin,  thus  : 

/OH 
CfiH  /  =         /OH 

\CHj.CHj.NH,.COOH      CgH/  +  COj. 

\CH2.CH2.NHj 

In  1883  Erlenmeyer  and  Lipp  observed  that  phenyl-a-amido- 
propionic  acid  (phenyl-alanin),  on  dry  distillation,  decomposed  with 
the  formation,  among  other  products,  of  a  base  having  the  composi- 
tion CgHjjN.  This  base  was  found  to  be  identical  with  phenyl- 
ethylamin,  CgHj.CHg.CHg.NHg,  and  in  its  properties  and  composition 
it  resembles  Nencki's  base.  Later  (1889),  Nencki  adopted  a  similar 
view  in  regard  to  the  nature  of  this  base,  and  regarded  it  as  possess- 
ing the  formula  just  given — that  is  phenyl-ethylamin.  He  con- 
sidered phenyl  amido-propionic  acid — one  of  the  three  aromatic 
nuclei  contained  in  the  albumin  molecule — as  the  source  of  this  base. 
From  the  fact  that  phenyl-a-amido-propionic  acid  is  a  well-known 
putrefactive  product,  it  would  seem  that  Nencki's  base  may  arise 
either  by  the  putrefactive  decomposition  of  that  acid,  or  by  the  hyd- 
rolytic  cleavage  of  the  acid  as  a  consequence  of  the  method  employed 
in  isolating  the  base.  The  latter  would  seem  to  be  the  most  prob- 
able explanation  of  the  genesis  of  this  base,  inasmuch  as  Brieger,  by 
using  his  method  for  the  isolation  of  ptomai'ns,  was  not  able  to  ob- 
tain it  from  putrid  gelatin. 

The  platinochlorid,  (CgHjiN.HCl)2PtCl,  (Pt  =  29.89  per  cent.),  is 
readily  soluble  in  hot,  and  but  slightly  soluble  in  cold  water,  and 
can  be  therefore  recrystallized  from  water.  It  forms  beautiful  flat 
needles.  On  dry  heating  it  gives  off  an  oil  which  possesses  an  odor 
resembling  very  much  that  of  xylol  or  cumol,  and  burns  with  a 
smoky  flame.  This  distinguishes  Nencki's  base  from  collidin,  since 
the  platinochlorid  of  the  latter  does  not  show  this  behavior. 

Nencki  also  obtained  from  putrid  gelatin,  under  certain  ill-defined 
conditions,  especially  when  no  glycocoll  was  present,  a  basic  product 
which  gave,  with  sulphuric  acid,  large  lamellar  crystals.  The  free 
base  forms  a  thick  colorless  syrup,  possessing  a  nauseous,  bitter  taste. 
It  did  not  become  crystalline  even  after  standing  some  time.  Unlike 
the  base  CgHj^N,  it  is  not  volatile,  and  is,  therefore,  obtained  on 
evaporation  of  the  acidulated  solution  after  previous  removal  of  the 
volatile  bases  by  distillation  with  baryta. 

A  Base,  CgHjjN,  isomer  of  collidin  and  of  the  preceding  base, 
with  which  it  is  possibly  identical,  was  obtained  by  O.  de  Coninck 
(1888)  in  the  later  stages  of  putrefaction  of  sea-polyps  (poulpes 
marins).     It  forms  a  yellowish,  rather  mobile  liquid,  possessing  a 


COLLIDIN  ISOMER.  257 

strong  benumbing  {yireuse)  odor,  and  is  but  slightly  soluble  in  water. 
It  is  soluble  in  methyl  and  ethyl  alcohol,  ether  and  acetone.  Its 
density  is  0.9865.  When  dried  over  potash  it  boils  at  202° 
without  undergoing  decomposition.  On  exposure  to  the  air  it  be- 
comes brown,  hydrates  rapidly,  and  the  boiling  point  is  then  lowered. 
It  has  not  been  noticed  to  absorb  carbonic  acid  from  the  air.  It 
resembles  some  of  the  bases  obtained  from  Dippel's  oil.  The  salts 
are  in  general  less  stable  than  those  of  the  pyridin  bases,  and  in  this 
respect  it  approaches  the  dihydro-pyridin  bases. 

On  treatment  with  hydrogen  peroxid  it  yields  an  isomer  of  my- 
din,  CgHjjNO.  The  simple  and  double  salts  of  this  hydroxyl  deriv- 
ative which  de  Coninck  (1899)  calls  collidone  are  decomposed  by 
boiling  water. 

The  hydrochlorid,  CglljjN.HCl,  forms  white  or  slightly  yellowish 
radiate  masses  which  are  deliquescent  and  very  soluble  in  water. 
The  hydrobromid,  CgH,jN.HBr,  resembles  it  but  is  less  deliquescent 
and  a  trifle  less  soluble  in  cold  water. 

The  platinochlorid,  (CgIIjjN.IICl)2PtCl^,  is  a  dark  orange-colored 
powder  which  is  insoluble,  or  almost  so  in  cold  water,  and  is  a 
rather  stable  compound.  Boiling  water  and  water  at  80°  decom- 
pose it  into  hydrochloric  acid  and  (CgHjjNCl)2PtCl2  which  is  a 
light-brown  powder,  insoluble  in  cold,  scarcely  soluble  in  hot  water. 

The  aurochlorid,  CglljjN.HCl.AuClg,  forms  a  light-yellow  pre- 
cipitate. It  is  quite  stable  in  cold,  but  very  unstable  in  hot  or  even 
warm  water.  It  cannot  be  modified  by  withdrawal  of  hydrochloric 
acid. 

The  base  forms  two  compounds  with  mercuric  chlorid.  (CgHj^N. 
IICl)2HgCl2  crystallizes  in  small  white  needles,  which  are  slightly 
soluble  in  water  and  in  dilute  alcohol,  insoluble  in  absolute  alcohol, 
and  on  exposure  to  moist  air  undergo  change.  The  second  com- 
pound, 2(CgHjjN.IICl).3HgCl2,  is  obtained  by  adding  an  excess  of 
concentrated  mercuric  chlorid  to  a  concentrated  solution  of  the  hydro- 
chlorid. It  forms  slightly  yellow,  somewhat  longer  needles  which 
are  insoluble  in  the  principal  solvents,  and  are  likewise  changed  by 
atmospheric  humidity. 

The  iodomethylate,  CgH^jN.CHgl,  is  formed  by  mixing  solu- 
tions of  the  base  and  methyl  iodid  in  absolute  ether.  It  is  deposited 
as  a  network  of  fine  white  needles,  which  are  but  slowly  altered  in 
the  air,  and  are  soluble  in  absolute  alcohol.  This  solution  on  the 
addition  of  a  little  potash  assumes  a  dark-red  color,  which  is  height- 
ened by  the  addition  of  a  little  hydrochloric  or  acetic  acid,  and  de- 
stroyed by  ammonia  without  any  resultant  fluorescence.  Warmed 
with  excess  of  moist  solid  potash  it  becomes  garnet-red  in  color  and 
gives  oif  an  odor  resembling  that  of  the  dihydropyridins.  It  thus 
behaves  the  same  as  the  pyridin  iodomethylates. 

On  oxidation  with  potassium  permanganate  it  yields  an  acid  which 
17 


258  CHEMISTRY  OF  THE  PTOMAINS. 

melts  at  229°-230°,  and  begins  to  sublime  at  150°.  It  presents  all 
the  characteristics  of  nicotinic  acid,  CgH^NOj,  which  is  formed  as 
the  result  of  oxidation  of  nicotin.  With  hydrochloric  acid  it  forms 
the  compound  CgH^NOg-HCl.  With  copper  acetate  it  forms  a 
salt ;  this  distilled  with  lime,  yields  a  substance  which  on  boiling 
with  platinum  chlorid  and  water  forms  the  compound  (C5H5NC1)2. 
PtClg.  This  same  substance  forms  an  iodomethylate,  which  in  al- 
coholic solution  gives,  on  addition  of  potash,  the  characteristic  re- 
action of  pyridin  bases. 

The  base  CgH^jN,  therefore,  yields  pyridin  and  nicotinic  acid. 

A  Base,  CgH^gN,  was  obtained  by  Gautier  and  Etard  (1881)  from 
the  chloroform  extracts  (see  method,  page  234)  from  putrefying 
mackerel,  as  well  as  from  the  decomposing  flesh  of  the  horse  and  ox. 
It  is  regarded  by  these  authors  as  a  constant  and  definite  product  of 
the  bacterial  fermentation  of  albuminoid  substances,  but  this  view  is 
hardly  justifiable,  inasmuch  as  the  base  has  not  been  found  by  other 
investigators.  It  is  accompanied  by  the  base  C^^IIggN^.  Nencki 
(1882)  asserted  the  identity  of  this  base  with  the  one  which  he  had 
isolated  in  1876,  and  to  which  he  had  ascribed  the  formula  CgHj^N. 
On  the  other  hand,  Gautier  and  Etard  consider  their  base  to  be 
identical  with  the  hydrocollidin  obtained  by  Cahours  and  Etard  by 
the  action  of  selenium  on  nicotin. 

An  isomer  of  this  base,  hemopyrrol,  has  been  prepared  from  hemin 
and  also  from  the  chlorophyll  derivative  phyllocyanin.  On  oxida- 
tion it  yields  urobilin  (Nencki  and  Marchlewski,  Ber.  d.  d.  chem. 
Ges.,  34,  1687). 

The  free  base  is  an  alkaline,  almost  colorless,  oily  liquid,  pos- 
sessing a  penetrating  odor  resembling  that  of  syringa.  It  is  volatile 
without  decomposition,  and  boils  at  about  205°,  while  hydrocollidin 
boils  at  210°.  Its  density  at  zero  is  1.0296.  When  exposed  to  the 
air  it  oxidizes  slowly,  becomes  brown  and  viscous,  and  at  the  same 
time  absorbs  carbonic  acid.  It  differs  from  a  collidin  in  possessing 
a  strong  reducing  action,  since  both  the  gold  and  platinum  double 
salts  become  reduced  on  heating,  and  even  in  the  cold. 

The  hydrochlorid,  CgHjgN.HCl,  is  very  soluble  in  water  and  in 
alcohol,  and  usually  forms  fine  needles  resembling  snow  crystals.  It 
is  neutral  in  reaction  and  possesses  a  bitter  taste.  In  the  presence 
of  an  excess  of  acid  it  reddens  and  resinifies.  f^^i 

The  platinochlorid,  (CgH,3N.HCl)2PtCl,  (Pt  =  29.7  per  cent.);' 
is  of  a  light-yellow,  flesh  color,  crystalline,  and  but  slightly  soluble. 
It  dissolves  on  warming,  and  recrystallizes  in  bent  needles. 

The  aurochlorid  is  rather  soluble,  and  becomes  slowly  reduced  in 
the  cold  ;  rapidly  on  warming. 

This  isomer  of  hydrocollidin  is  strongly  poisonous.  Even  so 
small  a  dose  as  0.0017  gram  of  the  hydrochlorid  produced,  when  in- 


PAEVOLIN  ISOMER.  259 

jected  under  the  skin  of  a  bird,  marked  unsteadiness  of  gait,  followed 
by  paralysis  of  the  extremities,  and  finally  death.  The  pupils  are 
normal  and  the  heart  stops  in  diastole.  Larger  doses  (0.007  gram) 
cause  at  first  vomiting  and  staggering,  which  soon  give  way  to  a 
condition  of  exaltation.  Toward  the  end  tetanic  convulsions  set  in, 
followed  by  almost  complete  paralysis. 

A  Base,  CgHjgN,  isomeric  with  parvolin,  has  been  extracted  by 
Gautier  and  Etard  (1881)  from  decomposing  mackerel  and  horse- 
flesh. The  method  employed  by  these  chemists  for  its  isolation  is 
given  on  page  233.  The  identity  of  this  base  with  the  synthetic 
parvolin,  obtained  by  Waage  by  heating  ammonia  with  propionic 
aldehyd  in  a  sealed  tube  at  200°,  cannot  be  considered  to  be  defi- 
nitely settled,  although  an  apparent  identity  exists  in  regard  to  their 
boiling  points.  Thus,  the  synthetic  parvolin  boils  at  193''-196°, 
while  Gautier  and  Etard  assign  to  their  base  a  boiling-point  a  little 
below  200°.  Further  investigation  is  necessary  to  decide  upon  the 
question  of  the  identity  of  this  base  with  parvolin,  or  of  the  ptomain 
CgHjgN  with  hydrocollidin. 

The  free  base  is  an  oily,  amber-colored  liquid,  possessing  the  odor 
of  hawthorn  blossoms.  It  is  slightly  soluble  in  water ;  very  soluble 
in  alcohol,  in  ether,  and  in  chloroform.  Its  boiling-point,  as  stated 
above,  is  a  trifle  below  200°.  Like  the  bases  CgHjgN  and  CjoH^gN 
it  becomes  brown  and  soon  resinifies  on  exposure  to  air. 

The  platinochlorid,  (CgHj3N.HCl)2PtCl,  (Pt  =  28.65  per  cent.), 
is  slightly  soluble,  crystalline,  and  flesh  colored ;  exposed  to  the  air 
it  soon  becomes  pink. 

The  aurochlorid  is  quite  soluble. 

A  Base,  Cj^Hj^N,  was  isolated  by  Guareschi  and  Mosso  (1883) 
from  ox-blood  fibrin  which  had  been  allowed  to  putrefy  for  five 
months.  In  1887  it  was  re-obtained  from  putrid  fibrin  by  Guares- 
chi, who  this  time  ascribed  to  it  the  formula  CjqHjjN.  In  1886 
Oechsner  de  Coninck  found  it  among  the  basic  products  formed  in 
the  putrefaction  of  the  jelly-fish  (^poulpes  marins,  Hugounenq,  page 
21).  The  method  used  for  its  extraction  was  that  of  Gautier  and 
Etard  (see  page  233).  It  forms  a  brownish  oil  of  strong  alkaline 
reaction,  which  soon  resinifies.  It  possesses  an  unpleasant,  weak 
pyridin  or  coniin  odor,  and  is  but  slightly  soluble  in  water ;  soluble 
in  ether  and  in  chloroform. 

In  regard  to  the  constitution  of  this  ptomain  we  know  nothing, 
but  from  its  physical  characters  it  would  seem  to  possess  a  pyridin 
nucleus.  It  is  isomeric  with  corindin,  a  homologue  of  parvolin  and 
collidin,  obtained  from  coal-tar. 

For  the  behavior  of  the  hydrochlorid  to  alkaloidal  reagents,  see 
Table  I. 


260  CHEMISTRY   OF  THE  PTOMAINS. 

The  hydrochlorid,  C^pHj^N-HCl,  crystallizes  in  colorless  choles- 
terin-like  plates  which  are  somewhat  deliquescent. 

The  platinochlorid,  (CioH^,N.HCl)2PtCl,  (Pt  =  27.52  per  cent.), 
forms  a  light  flesh-colored,  crystalline  precipitate,  and  is  insoluble 
in  water,  alcohol  and  ether.  It  does  not  resinify,  and  is  stable  at 
100°. 

In  its  action  this  ptomain  resembles  curara,  although  it  is  by  no 
means  as  strong.  0.012  gram  of  the  free  base  produced  in  a  frog 
dilatation  of  the  pupil  and  slowing  of  the  respiration.  The  nostrils 
were  motionless,  and  within  five  hours  complete  paralysis  of  the 
muscles  took  place.  The  reflex  excitability  gradually  diminished 
until  it  finally  disappeared.  An  orange-blossom  odor  was  observed 
about  the  frogs  which  were  poisoned  by  this  ptomain.  The  same 
amount  of  ptomain  injected  into  a  greenfinch  produced  vomiting, 
and  a  condition  of  weakness  and  decreased  sensibility,  followed  soon, 
however,  by  recovery.  A  rat  was  not  affected  by  0.020  gram  of  the 
free  base.     The  hydrochlorid  acts  much  more  energetically. 

A  Base,  C^^Hj^N,  was  isolated  by  O.  de  Coninck,  in  1886  (Hu- 
gounenq,  page  21,  C  Rendus,  1888),  from  sea-polyps  in  an  advanced 
stage  of  putrefaction,  together  with  the  base  CgH^^N.  The  method 
employed  for  its  extraction  was  that  of  Gautier  and  Etard  (see  page 
233).  Nesbitt  (1899)  obtained  a  similar,  if  not  identical,  base  from 
the  intestinal  contents  of  a  dog  after  previous  ligature  of  the  lower 
part  of  the  intestine.  It  was  associated  with  cholin  and  neurin.  It 
forms  a  slightly  yellow,  viscous  liquid,  and  possesses  a  pleasant  odor 
resembling  that  of  blooming  broom.  Its  density  is  about  1.18.  It 
boils  at  about  230°  (uncorrected),  with  initial  decomposition.  In 
water  it  is  but  slightly  soluble,  readily  so  in  ether,  alcohol,  acetone, 
and  ligroin.  It  is  rapidly  oxidized  by  the  air,  becomes  brown,  and 
resinifies  but  does  not  absorb  carbonic  acid. 

The  hydrochlorid,  CjgHjgN.HCl,  forms  fine  yellowish,  very  deli- 
quescent needles  which  in  the  presence  of  a  trace  of  air  are  at  once 
colored  red ;  if  more  air  is  present  the  red  changes  to  a  brown,  and 
in  the  open  air  a  resin  is  formed  the  same  as  from  the  free  base.  It 
is  very  easily  soluble. 

The  hydrobromid,  CjpHj^N.HBr,  crystallizes  in  a  network  of 
fine  deliquescent  needles  which  become  likewise  red  on  exposure  to 
air.  It  is  very  soluble  in  water ;  less  so  in  strong  alcohol,  and 
almost  insoluble  in  ether. 

The  platinochlorid,  (CjQlIj,N.HCl)2PtCl^,  forms  a  dark-red  pow- 
der which  is  insoluble  in  cold  water ;  very  soluble  in  warm  water. 
It  can  be  kept  in  dry  air ;  in  moist  air,  it  loses  hydrochloric  acid 
and  becomes  partially  oxidized.  Boiling  water  decomposes  it. 
(Cj|,H^5N.Cl)2PtCl2  forms  clear  brown  plates  which  are  stable  in 
moist  air,  and  melt  at  206°.    It  is  insoluble  in  cold  water,  soluble  in 


A  BASE.  261 

boiling  water,  but  decomposes.  In  recrystallizing,  warm,  previously 
boiled  water  should  be  used. 

The  aurochlorid,  CjQHj.N.HCl.AuClg,  occurs  as  a  light  yellow 
precipitate  ;  insoluble  in  cold  water,  soluble  in  warm  water.  It  is 
decomposed  by  boiling  water ;  is  stable  when  kept  in  a  moist  atmos- 
phere. 

The  iodomethylate,  CjQHj^N.CHgl,  in  warm  alcoholic  solution 
yields,  on  the  addition  of  strong  potash,  a  bright  red  color,  which 
soon  becomes  brown,  and  in  about  an  hour  the  solution  shows  a 
greenish-blue  fluorescence.  This  rapidity  of  change  is  due  to  the 
extreme  oxidizability  of  the  ptomain. 

On  careful  oxidation  with  potassium  permanganate  at  ordinary 
temperature  it  yields  a  solid  acid,  having  a  melting-point  of  228°— 
229°,  the  same  as  that  of  the  pyridin  carbonic  acid  of  Huber  and 
Laidlin,  obtained  by  the  oxidation  of  nicotin.  The  solubility  in 
cold  and  in  warm  water  and  in  absolute  alcohol  is  the  same  as  that 
of  nicotinic  acid.  It  begins  to  sublime  at  150°  as  pearly  spangles. 
The  formula  is  CgH^NOj.  On  distillation  with  lime  pyridin  forms. 
It  is,  therefore,  identical  with  nicotinic  acid,  an  oxidation  product 
of  nicotin  and  other  volatile  alkaloids. 

O.  de  Coninck  considers  this  base,  as  well  as  CgHjjN,  as  belong- 
ing to  the  pyridin,  and  not  to  the  hydropyridin  series. 

A  Base,  Cj^Hj^N,  was  described  by  Griffiths  (1890)  as  derived 
from  cultures  on  pepton-agar  of  the  bacterium  allii,  a  germ  obtained 
from  putrid  onions.  The  base  (hydrochlorid  ?)  forms  colorless,  pris- 
matic, microscopic,  very  deliquescent  needles,  which  are  soluble  in 
warm  water,  alcohol,  ether  and  chloroform.  It  gives  a  hawthorn- 
like odor,  especially  when  warmed.  With  phosphomolybdic  acid  it 
yields  a  white  ;  with  iodin  in  potassium  iodid  and  with  tannic  acid  a 
chestnut-colored  precipitate.  Nessler's  solution  produces  a  yellow 
chestnut-colored  precipitate.  Picric  acid  throws  down  a  yellow 
slightly  soluble  deposit.  The  platinochlorid,  (CioHj^N.HCl)2PtCl4, 
is  yellow,  crystalline,  and  difficultly  soluble  in  cold  water  and  in 
alcohol ;  soluble  in  warm  water.  Gold  chlorid  produces  a  thick  yel- 
low precipitate  soluble  in  water.  Dilute  sulphuric  acid  produces  a 
violet-red  color.     The  base  is  apparently  a  hydrocoridin. 

Griffiths  has  also  described,  together  with  a  variety  of  other  prod- 
ucts in  physiological  chemistry,  the  basic  compounds  which  are  given 
in  the  subjoined  table.  The  method  employed  in  the  isolation  of  all 
but  few  of  these  compounds  is  that  of  Luff  (see  next  chapter  under 
urine). 

Bacillus  allii,  QoH^N  1890. 

Scarlet-fever  urine,  Scarlatinin,  C5H12NO4  1891. 

Diphtheria        "  Diphtherin,  CuHi7N30e  " 

Parotitis  "  Propyl-glycocyamin,  CjHjjNsO^  " 


Glanders   urine, 

CijHioN.Oe 

Pneumonia   " 

CjoHseN.Os 

Measles         " 

Glycocyamidin, 

C3H6N3O 

Whooping  cough  urine, 

CsHigNO, 

Erysipelas                  ' ' 

Erysipelin, 

CUH13NO3 

Puerperal  fever        " 

Puerperalin, 

Cj^HigNO, 

Epilepsy                   ' ' 

Ci^H^eNsO, 

Micrococcus  tetragenus, 

CsHeNO, 

Bacillus  pluviatilis, 

CgH^iN^Oa 

Eczema  urine, 

Eczemin, 

C,H,5N0 

Influenza    " 

CgHgNO^ 

Putrid  sardines, 

Sardinin, 

Cx,HiiNO, 

Cancer  urine, 

Cancerin, 

CsH^NOs 

Pleurisy    ' ' 

Pleuricin, 

C5H,0, 

Angina  pectoris  urine, 

C10H9NO, 

262  CHEMISTRY  OF  THE  PTOMAINS. 

1892. 
(I 

<( 

ia92. 

(< 
(( 
<< 
li 
(I 

1893. 

<( 

(( 

1894. 

<( 

1895. 

It  is  very  suspicious  to  find  such  a  long  array  of  products,  isolated 
apparently  with  the  greatest  ease  by  a  most  simple  method.  This 
is  all  the  more  remarkable  in  view  of  the  fact  that  other  skilled 
workers  have  utterly  failed  in  some  of  these  cases  to  isolate  basic 
substances.  Thus,  cultures  of  Loeffler's  bacillus  of  diphtheria  have 
been  shown  by  Brieger  and  Frankel  and  others  not  to  contain 
ptomains,  and  yet  Griffiths  claims  to  isolate  a  base  from  the  urine  in 
the  disease,  and  also  from  the  pure  cultures  of  the  bacillus.  The 
base  is  apparently  so  abundant  that  in  1893  he  was  able  to  sacrifice 
1.5  g.  (!)  in  order  to  show  that  a  disinfectant  *'izal"  destroyed  its 
poisonous  properties  (!).  Again,  from  four  gallons  of  scarlet 
fever  urine  Luff  succeeded  in  obtaining  some  crystals  of  a  base, 
insufficient  however,  for  analysis.  Griffiths,  however,  not  only 
isolated  it  in  sufficient  quantity,  but  had  2.5  g.  of  it  to  spare 
for  testing  the  power  of  "  izal."  Further  than  that,  he  succeeded 
in  isolating  scarlatinin  from  pure  cultures  of  the  micrococcus 
scarlatinas  (!).  We  have  yet  to  learn  that  scarlet  fever  is  due 
to  a  micrococcus. 

Equally  interesting  facts  appear  in  connection  with  the  base 
of  glanders,  which  was  isolated  from  the  urine  and  also  from 
pure  cultures  of  the  glanders  bacillus.  Nencki,  however,  had  10 
liters  of  a  bouillon  culture  of  the  glanders  bacillus  examined  ac- 
cording to  Griffiths's  method,  and  failed  to  obtain  a  weighable 
quantity  of  a  ptomain  (Maly's  JaJu'esbericht,  1894,  24,  601).  The 
action  of  this  basic  product  of  glanders  is  remarkable,  producing 
"  an  abscess  at  the  point  of  inoculation,  nodules  in  the  lungs  and 
spleen,  and  metastatic  abscesses  in  various  organs." 

The  description  of  these  bases  is  so  brief  and  unsatisfactory  that 
the  description  of  one  applies  almost  equally  well  to  all  the  others. 
Thus  the  bases  are  all  white,  crystalline,  and  soluble  in  water,  im- 
parting an  alkaline  reaction  in  all  but  two  or  three  cases.  All  but 
four  or  five  are  characterized  as  poisonous,  but  the  dose  employed  is 
never  given ;  the  method  of  administration  is  mentioned  but  twice,  and 
the  kind  of  animal  employed  but  six  times.     All,  or  nearly  all,  form 


ETHYLIDENEDIAMIN.  263 

crystalline  hydrochlorids,  aurochlorids,  and  platinochlorids.  Reac- 
tions are  usually  given  with  only  three  or  four  reagents.  It  will  be 
noticed  that  one  of  these  products  has  no  nitrogen  and  yet  is  men- 
tioned as  a  ptomain  (!) ;  further  that  the  formula  of  five  of  these 
compounds  is  not  in  accord  with  the  law  of  even  numbers.  Consid- 
ering the  amounts  of  the  bases  available  for  "  izal "  experiments,  it 
is  proper  to  expect  accurate,  exhaustive,  thorough  work.  Chemical 
science  is  not  advanced  by  coining  names  or  establishing  formulae. 

A  Base,  CgjHgjN,  was  obtained  by  Delezinier  (1889)  and  is  said 
to  be  the  alkaloid  isolated  in  1879  by  Brouardel,  which  in  its  chem- 
ical and  physiological  properties  was  described  as  similar  to  veratrin. 
It  forms  an  almost  colorless  oily  fluid,  which  possesses  a  hawthorn- 
like odor.  It  is  very  readily  oxidizable  and  yields  the  veratrin-like 
reactions  only  in  the  presence  of  air.  It  is  soluble  in  alcohol,  ether, 
toluene  and  benzene ;  and  forms  well-defined  salts  which  are  very 
deliquescent.  It  appears  to  be  an  amin  and  in  its  composition  dif- 
fers from  cevadiu  by  911^0.  Nothing  is  stated  in  regard  to  its 
source  or  method  of  preparation.  The  analytical  results  given 
(C  =  89.41,  H  =  7.3,  N  =  3.03)  correspond  more  to  the  formula 
C3.H33N. 

Ethylidenediamin  (?),  C2HgN2. — This  base  was  considered  at  first 
by  Brieger  to  be  identical  with  ethylenediamin,  but  subsequent  com- 
parison showed  this  to  be  an  error.  Thus  the  former  is  poisonous 
and  does  not  form  a  gold  salt,  while  the  latter  is  not  poisonous  and 
does  form  a  rather  difficultly  soluble  aurochlorid.  Again,  ethylenedi- 
amin forms  a  platinochlorid  which  is  almost  insoluble  in  hot  water, 
whereas  the  platinum  double  salt  of  the  ptomain  is  much  more  easily 
soluble.  Brieger,  therefore,  inclined  to  the  belief  that  it  was  iden- 
tical with  ethylidenediamin,  CH3.CII(NH2)2,  rather  than  with  ethyl- 
enediamin, which  has  the  structure  CH2.NII2.CII2.NH2.  This 
ptomain  was  obtained  by  Brieger,  1885  (I.,  44),  from  decomposing 
haddock  (see  gadinin).  Kulneif  has  probably  met  wdth  this  base  in 
the  liquids  of  the  stomach  in  gastrectasis.  Carbone  has  reported  it 
in  cultures  of  the  proteus  vulgaris  with  gadinin,  trimethylamin,  and 
cholin. 

The  free  base  can  be  obtained  without  decomposition,  on  distilling 
the  hydrochlorid  with  sodium  hydrate. 

The  hydrochlorid,  C2HgN2.2HCl,  crystallizes  in  long,  glistening 
needles  which  are  readily  soluble  in  water,  insoluble  in  absolute 
alcohol.  It  gives  no  combination  with  gold  chlorid.  For  its  be- 
havior to  alkaloidal  reagents  see  Table  I. 

The  platinochlorid,  C2H3N2.2HCl.PtCl,  (Pt  =  41.49  per  cent.), 
forms  small  yellow  plates  which  are  moderately  difficultly  soluble  in 
water.     It  can  be  readily  recrystallized  from  hot  water. 


264  CHEMISTRY  OF  THE  PTOMAINS. 

Frogs  seem  to  be  less  susceptible  to  the  action  of  this  poison  than 
mice  or  guinea-pigs.  In  the  latter,  it  produces  a  short  time  after 
injection  an  abundant  periodic  flow  of  secretion  from  the  nose,  mouth 
and  eyes.  The  pupils  dilate  and  the  eyeballs  project.  Violent 
dyspnoea  then  comes  on  and  predominates  until  the  death  of  the 
animal,  which  does  not  take  place  for  twenty-four  hours  or  more. 
The  heart  is  stopped  in  diastole. 

Pohl  (1898)  has  studied  the  effect  of  synthetic  ethylenediamin 
upon  animals.  The  subcutaneous  injection  of  1  gram  in  rabbits 
(IJ  k.)  produced  no  effect  until  after  the  lapse  of  a  day,  when  a 
serous  exudate  formed  at  the  point  of  inoculation.  Death  from 
parenchymatous  nephritis  followed  on  the  third  or  fourth  day. 
Larger  doses  produced  clonic  convulsions  and  paralysis  of  the  ex- 
tremities. In  dogs,  subcutaneous  injections  had  no  effect,  but  in- 
travenously the  diamin  proved  fatal. 

Trimethylenediamin  (?),  CgHj^Ng  (?),  is  a  toxic  base  isolated  by 
Brieger  (1887)  from  cultures  of  the  comma  bacillus  on  beef-broth. 
It  may  be  stated  here  that  from  the  same  source,  cholera  cultures, 
Kunz  (1888)  obtained  a  base  which  he  considered  to  be  identical 
with  spermin  or  ethyleneimin  (see  next  chapter).  It  is  present, 
however,  in  exceedingly  minute  quantity,  and  occurs  in  the  mercuric 
chlorid  precipitate,  from  which  it  is  obtained  by  the  following 
method  :  The  precipitate  is  decomposed  by  hydrogen  sulphid,  the 
filtrate  evaporated  to  dryness,  and  the  residue  taken  up  with  abso- 
lute alcohol  and  precipitated  by  an  alcoholic  solution  of  sodium 
picrate.  The  precipitate  thus  obtained  consists  of  the  picrates  of 
cadaverin,  creatinin,  and  of  this  new  base.  It  is  boiled  with  abso- 
lute alcohol  to  remove  the  insoluble  cadaverin  picrate ;  the  filtrate  is 
evaporated  to  expel  the  alcohol,  and  the  bases  then  converted  into  the 
platinum  double  salts,  whereby  the  easily  soluble  creatinin  platino- 
chlorid  can  be  separated  from  the  corresponding  less  soluble  com- 
pound of  the  new  base. 

Owing  to  the  small  quantity  of  this  substance  present,  a  complete 
study  of  its  properties  has  not  as  yet  been  made.  It  gives  difficultly 
soluble  precipitates  with  gold  chlorid  and  with  platinum  chlorid  ; 
the  compound  with  the  latter  crystallizes  in  long  needles.  With 
picric  acid  it  gives  a  precipitate  consisting  of  felted  needles,  which 
resemble  creatinin  picrate;  they  melt  at  198°.  Phosphomolybdic 
acid  yields  a  precipitate  crystallizing  in  plates,  while  potassium  bis- 
muth iodid  gives  dark-colored  fine  needles. 

In  its  physiological  action  it  seems  to  be  identical  with  the  basic 
substance  isolated  from  choleraic  bodies  by  different  observers.  It 
causes  violent  convulsions  and  muscle-tremor.  The  action  of  the 
synthetic  base  has  also  been  studied  by  Pohl  and  apparently  it  is 
more  toxic  than  the  preceding.     The  action  of  the  heart  is  slowed 


PUTRESCIN.  265 

and  irregular ;  the  pulse  is  feeble,  respiration  is  slow  and  deep  and 
marked  dyspnoea  prevails.  These  symptoms  soon  disappear  but  the 
rabbit  died  in  about  four  days  with  ursemic  symptoms.  Albumi- 
nuria was  marked. 

Besides  trimethylenediamin  another  toxin  was  obtained  by  Brieger 
from  cholera  cultures,  but  in  quantity  insufficient  for  analysis.  It 
was  obtained  from  the  mercuric  chlorid  filtrate  after  elimination  of 
methylamin,  trimethylamin,  and  traces  of  cholin  and  creatinin,  as  an 
insoluble  platinum  double  salt.  Subcutaneous  injection  of  this  base 
into  mice  produced  a  paralysis-like  lethargic  condition,  slowing  of 
respiration  and  heart's  action,  lowering  of  temperature,  and,  finally, 
death  in  twelve  to  twenty-four  hours.  In  some  cases  bloody  stools 
were  passed. 

Putrescin,  C4HJ2N2 ,  is  a  diamin  which  almost  invariably  occurs 
together  with  cadaverin  with  which  it  is  closely  related.  This  base 
was  also  discovered  by  Brieger  in  1885  (II.,  42),  who  obtained 
it  from  putrefying  human  internal  organs  (for  four  months  at  a  low 
temperature  without  access  of  much  oxygen) ;  and  from  the  same 
material  decomposing  at  the  ordinary  temperature  of  the  room  for 
from  three  days  to  three  weeks.  It  has  also  been  obtained  from 
herring,  twelve  days  in  spring  ;  from  pike,  six  days  in  summer  ;  from 
haddock,  two  months  (Bocklisch).  Also  from  putrid  mussel,  sixteen 
days  (Brieger) ;  and  from  human  as  well  as  horseflesh.  Brieger  like- 
wise obtained  it  from  cultures  of  the  bacteria  of  human  feces  on  gel- 
atin, and  in  small  quantity  in  rather  old  cultures  of  the  comma  bacil- 
lus on  beef-broth  ;  in  larger  quantity  in  cultures  of  the  same  germ  on 
blood-serum.  Garcia  found  it  in  putrefying  meat  and  pancreas, 
together  with  cadaverin  and  hexamethylenediamin.  The  diamin 
production  at  30°  is  considerable  in  twenty-four  hours  ;  reaches  its 
maximum  in  three  days.  Putrescin  appears  on  the  first  day.  Roos 
found  putrescin  in  stools  of  one  case  of  cholera  and  in  two  cases  of 
diarrhoea  or  cholerin.  With  cadaverin  it  forms  in  the  sterile  auto- 
digestion  of  pig's  stomach  (Lawrow). 

Udranszky  and  Baumann  in  1888  demonstrated  the  existence  of 
putrescin  and  cadaverin  in  the  urine  of  cystinuria.  They  found  the 
total  amount  of  the  dibenzoyl  compounds  in  the  urine  in  1888  to 
vary  from  0.2-0.4  g.  per  day.  Cadaverin  made  up  about  f  of  this 
amount,  and  putrescin  ^  -  5.  Garcia  examined  the  same  patient  in 
1892  and  obtained,  as  an  average  of  seven  days,  only  0.064  g.  of 
the  dibenzoyl-compound,  which  contained  no  cadaverin  (!),  only 
putrescin.  With  ordinary  diet  the  average  of  11  days  was  0.027  g.; 
with  cheese-diet  an  average  of  8  days  gave  0.136  g.;  while  a  carbo- 
hydrate diet,  an  average  of  7  days,  gave  0.102  g.  of  the  dibenzoyl- 
compound.  In  the  feces  of  the  same  patient,  on  the  contrary, 
Udranszky  and  Baumann  found  in  1888  that  putrescin  constituted 


266  CHEMISTRY  OF  THE  PTOMAINS. 

by  far  the  greater  quantity,  while  cadaverin  formed  but  10  to  15 
per  cent.  Garcia,  in  1892,  showed  in  the  same  patient  the  presence 
of  only  putrescin  in  the  feces,  no  cadaverin.  The  feces  contained  on 
ordinary  diet,  an  average  of  11  days,  1.123  g.;  on  cheese-diet,  aver- 
age of  8  days,  1.3978  g.;  on  carbohydrate  diet,  average  of  7  days, 
0.741  g.  of  the  dibenzoyl  compound.  Borissow,  in  1894,  again  ex- 
amined the  feces  of  the  same  patient,  and  found  as  an  average  of 
four  days  2.062  g.  of  the  dibenzoyl-compounds,  which  contained 
only  traces  of  cadaverin.  It  would  seem  that  diet  and  the  in- 
tensity of  intestinal  decomposition  influence  the  amount  of  excretion 
of  diamins.  The  administration  of  salol  and  sulphur  (Mester), 
or  intestinal  lavage  (U.  and  B.),  had  no  effect  on  diamin  excretion. 
Normal  feces,  as  well  as  the  feces  in  various  diseases,  with  the  pos- 
sible exception  of  cholera  stools,  are  free  from  diamins.  It  would 
seem,  therefore,  that  these  bases  occur  in  cystinuria  as  the  result  of 
putrefactive  changes  going  on  in  the  intestines ;  becoming  partly 
absorbed  they  appear  in  the  urine  (see  p.  269).  In  two  cases  of 
cystinuria,  reported  by  Brieger  and  Stadthagen,  cadaverin  was 
found  almost  solely  present  in  the  urine. 

According  to  Mester,  the  diamins  are  proportionate  to  the  amount 
of  cystin  excreted,  and  therefore  constitute  a  fixed  symptom,  the 
cause  of  which  is  the  same  as  that  of  the  cystinuria. 

Although  putrescin  is  recognizable  on  about  the  fourth  day  of  the 
putrefaction,  yet  it  does  not  occur  in  appreciable  quantity  until 
about  the  eleventh  day.  The  amount  that  is  formed  increases  as  the 
putrefaction  goes  on,  so  that  a  considerable  quantity  may  be  obtained 
after  two  or  three  weeks.  A  very  good  source  for  the  preparation 
of  putrescin,  cadaverin,  and  neuridin  is  gelatin  which  has  been  al- 
lowed to  decompose  in  contact  with  water  for  some  weeks.  Neuridin 
is,  apparently,  formed  first,  but  is  soon  replaced  by  the  former  two 
bases.  In  the  process  of  extraction  it  is  first  obtained  in  the  alco- 
holic mercuric  chlorid  precipitate.  For  its  separation  from  cadaverin 
and  other  accompanying  bases,  see  saprin,  p.  278. 

From  the  urine  of  cystinuria  it  is  best  obtained  by  precipitation 
with  benzoyl  chlorid  (Baumanu's  method).  For  this  purpose  about 
1,500  c.c.  of  urine  are  treated  with  200  c.c.  of  sodium  hydrate  solu- 
tion (10  per  cent.),  then  20  to  25  c.c.  of  benzoyl  chlorid  are  added, 
and  the  whole  shaken  till  the  odor  of  the  latter  disappears.  The 
yellowish-white  precipitate  which  forms  may  consist  of  insoluble 
phosphates,  carbohydrates,  polyatomic  alcohols,  and  diamins.  The 
cystin  compound  is  precipitated  only  in  concentrated  solutions.  The 
precipitate  contains  from  a  half  to  two-thirds  of  the  diamins  present ; 
it  is  filtered  off,  digested  with  warm  alcohol,  and  the  solution 
filtered.  The  alcoholic  filtrate  is  concentrated  and  then  poured  into 
about  thirty  times  its  volume  of  cold  water.  The  diamin  compounds 
then  crystallize  out.     To  separate  the  two  diamins  they  are  redis- 


PUTRESCIN.  267 

solved  in  just  sufficient  warm  alcohol  to  effect  solution,  and  this  is 
then  poured  into  about  twenty  times  its  volume  of  ether.  The 
putrescin  benzoyl  compound  is  thus  thrown  out  of  solution.  The 
filtrate  from  this,  on  concentration,  yields  the  cadaverin  compound. 
To  isolate  that  portion  of  the  diamins  whicli  remains  in  the  original 
filtrate  with  benzoyl-cystin,  it  is  acidulated  with  sulphuric  acid  and 
extracted  with  ether.  The  residue  obtained  on  evaporating  the 
ethereal  solution  is  first  neutralized  with  a  12  per  cent,  sodium 
hydrate  solution,  then  mixed  with  three  to  four  times  its  volume  of 
the  same  solution.  The  precipitate  which  forms  consists  of  the 
sodium  compounds  of  benzoyl-cystin  and  the  diamins.  It  is  washed 
with  sodium  hydrate,  and  the  two  compounds  separated  by  their 
different  solubilities  in  water — the  cystin  compound  is  readily 
soluble,  that  of  the  diamins  insoluble.  To  purify  the  benzoyl- 
diamins  they  are  dissolved  in  warm  alcohol  and  precipitated  with 
excess  of  water. 

Putrescin  (from  putrescere,  to  rot,  to  putrefy)  is  a  water-clear,  rather 
thin  liquid  w^hich  fumes  in  the  air  and  has  a  peculiar  semen-like 
odor,  almost  undistinguishable  from  that  of  cadaverin  and  remind- 
ing one  somewhat  of  the  pyridin  bases.  It  absorbs  carbonic  acid 
energetically  from  the  air,  without  losing  thereby  the  repulsive  odor. 
The  boiling  point  of  the  free  base,  as  ordinarily  obtained,  is  about 
135°.  It  is  not  decomposed  by  distillation  with  potassium  hydrate, 
and  is  rather  difficultly  volatile  with  steam.  With  acids  it  forms 
beautiful  crystalline  salts.  Putrescin  unites  with  water,  like  ethyl- 
enediamin,  to  form  a  hydrate,  and  this  water  can  only  be  removed 
by  distillation  over  metallic  sodium.  The  perfectly  anhydrous  base 
boils  at  156°-157°,  and  then  solidifies  to  plates  (Brieger),  which 
melt  at  24°  (Udranszky  and  Baumann).  The  synthetic  base  boils 
at  158°-160°,  and  melts  at  23°-24°  (Ladenburg).  Like  cadaverin 
it  is  difficultly  soluble  in  ether. 

The  constitution  of  putrescin  has  been  determined  by  Udranszky 
and  Baumann  (1888).  They  showed  that  the  dibenzoyl  compound 
of  putrescin  was  identical  with  that  of  the  synthetic  tetramethylene 
diamin  and  of  the  base  which  they  found  in  the  urine  of  cystinuria. 

Putrescin,  therefore,  is  tetramethylenediamin,  a  homologue  of 
cadaverin,  and  its  rational  formula  is  : 

NH^.  CHj.  CHj.  CHj.  CH,.  NH,. 

The  same  authors  (^Zeitschr.  f.  physiol.  Chem.,  13,  591)  pointed 
out  that  diamins  might  possibly  occur  in  putrefaction  as  the  result 
of  oxidation  of  monamins.  Thus,  putrescin  might  arise  from  methyl- 
amin  according  to  the  equation  : 

CHs.CHj.NH,  CHj.CH2.NH2 

+  0=  I  +H2O. 

CH,.CH,.NH,  CH,.CH,  NH, 


268  CHEMISTRY  OF  THE  PTOMAINS. 

In  a  similar  manner  cadaverin  might  form  from  ethyl  and  pro- 
pylamin.  It  is  well  known  that  in  the  decomposition  of  proteids 
in  the  presence  of  carbohydrates  no  aromatic  compounds,  as  indol, 
phenol,  tyrosin,  etc.,  form.  With  reference  to  the  formation  of 
diamine  Garcia  has  shown  that,  in  the  presence  of  cane  sugar,  putre- 
fying meat  and  pancreas  yield  from  one-half  to  less  then  one-tenth 
as  much  diamins  as  when  no  sugar  is  present.  A  similar  decrease 
of  diamins  in  cystinuria  is  observed  (page  265)  when  the  patient  is 
placed  on  a  carbohydrate  diet. 

The  researches  of  EUinger  (1898),  however,  have  definitely  estab- 
lished the  source  of  putrescin  and  of  its  homologue  cadaverin.  All 
proteids  contain  the  hexon  base  arginin  which  on  heating  with  baryta 
yields  urea  and  ornithin  (which  see).  On  subjecting  the  latter, 
which  is  di-amido  valerianic  acid,  to  putrefaction  EUinger  obtained 
putrescin.  In  like  manner  lysin,  another  hexon  base,  yielded  cada- 
verin. These  two  ptomains,  therefore,  result  from  the  cleavage  of  the 
di-amino  acids  which  exist  preformed  in  the  proteid  molecule.  The 
fact  that  gelatin  yields  over  10  per  cent,  of  these  acids  explains  the 
abundant  yield  of  putrescin  and  cadaverin  in  the  putrefaction  of 
this  material. 

The  relation  of  diamins  to  cystinuria  has  until  recently  been 
scarcely  understood.  Baumann  and  Udranszky,  accepting  the  intes- 
tinal origin  of  the  diamins,  supposed  that  these  bases  entered  into 
combination  with  cystin,  protecting  it  against  oxidation.  When  fed 
to  dogs,  however,  the  diamins  are  in  part  excreted  as  such,  but  no 
cystin  appears.  Again,  cystin  is  not  present  in  the  feces  of  cysti- 
nuria. Werigo  considered  cadaverin  as  a  normal  product  of  pan- 
creatic digestion.  Garcia  has  shown  that  in  meat  and  pancreas 
putrefaction  diamin  formation  begins  on  the  first  day,  and  reaches 
its  maximum  on  the  third  day.  Furthermore,  meat  and  pancreas 
flasks  inoculated  with  the  feces  of  a  cystinuric  patient  produced  an 
increased  formation  of  diamins,  thus  apparently  confirming  the  view 
that  diaminuria  is  the  result  of  the  activity  of  certain  bacteria  in  the 
intestines. 

It  is  possible  that  in  diaminuria  some  other  product  is  formed, 
which,  when  absorbed,  combines  with  cystin  and  protects  it  against 
oxidation,  so  that  it  appears  in  the  urine.  Diaminuria  and  cystinu- 
ria certainly  go  hand  in  hand.  Garcia  endeavors  to  account  for  the 
presence  of  diamins  in  cystinuria  by  the  supposition  that  cystin,  the 
normal  product  of  the  body,  can  undergo  reduction  and  yield  putrescin 
under  the  influence  of  special  intestinal  bacteria.  Cystin,  however, 
is  not  present  in  feces  in  cystinuria,  and  when  fed  to  dogs  it  merely 
serves  to  increase  the  amount  of  sulphuric  acid  eliminated.  Moreover, 
the  formula  of  cystin  hardly  permits  of  the  derivation  of  diamins. 

With  reference  to  cystin  it  may  be  well  to  note  that  until  recently 
it  has  been  met  with,  outside  of  cystinuria,  only  in  a  drunkard's 


PUTRESCIN.  269 

liver  (Scherer);  in  beef  kidneys  (Cloetta);  in  decomposing  pancreas 
(Kiilz);  in  the  liver  of  a  horse  and  a  dolphin  (Dreschel),  and  in  a 
tyrosin  preparation  from  horn  (Emmerling),  The  studies  of  Baumann 
and  of  Dreschel  led  these  investigators  to  regard  cystin  as  a  normal 
intermediate  waste-product,  but  no  definite  evidence  of  this  was 
brought  out  until  Morner  (1899)  succeeded  for  the  first  time  in  pre- 
paring cystin  by  the  hydrolysis  of  horn  (6.8  per  cent.),  egg-mem- 
brane (6  per  cent.),  and  human  hair  (12.6  per  cent.).  Embden 
(1901)  obtained  it  in  like  manner  from  serum  and  egg-albumin. 
Moreover,  both  confirmed  Kiilz's  observation  as  to  the  formation  of 
cystin  in  tryptic  digestion.  Apparently  all  proteids  which  contain 
sulphur  in  a  form  which  can  easily  be  split  off  contain  the  cystin 
group.  This  fact,  considered  in  connection  with  the  now  known 
source  of  the  diamins,  renders  it  evident  that  cystinuria  is  due  to  a 
condition  of  abnormal  tissue  metabolism  rather  than  to  a  peculiar 
intestinal  decomposition.  The  presence  of  diamins  in  the  intestine 
is  probably  due  to  an  elimination. 

PutrescLn  can  be  prepared  synthetically,  according  to  Ladenburg's 
method,  by  converting  ethylene  bromid  into  the  cyanid  and  then 
reducing  this  by  means  of  sodium  in  absolute  alcohol.  It  is  an 
isomer  of  Angeli's  dimethylethylenediamin. 

On  heating  the  concentrated  aqueous  solution  of  the  hydrochlorid 
with  potassium  nitrite  there  is  produced  an  oil,  soluble  in  water, 
from  which  it  can  be  extracted  with  ether.  This  oil,  on  treatment 
with  phenol  and  sulphuric  acid,  gives  Liebermann's  nitroso-reaction, 
which  would  seem  to  show  that  putrescin  is  not  a  primary  diamin 
(butylenediamin),  but  is  rather  a  secondary  diamin  (Brieger,  II.,  42). 
As  a  primary  diamin  it  should  take  up,  on  repeated  treatment  with 
methyl  iodid,  six  methyl  radicals ;  whereas,  if  it  is  a  secondary  dia- 
min, only  four  methyl  radicals  can  enter  the  molecule.  Thus,  to 
illustrate,  methy  lamin,  CH3.NH2  (a  primary  amin),  combines  with 
three  molecules  of  methyl  iodid  to  form  (CH3)^N.III.  Similarly, 
dim  ethy lamin,  (€113)2.  NH,  requires  only  two  molecules  to  form 
(€113)^^.111.  In  the  case  of  diamins,  double  this  number  of  methyl 
groups  is  required  to  effect  complete  saturation.  As  a  matter  of  fact, 
Brieger  (III.,  101),  on  treating  putrescin  with  methyl  iodid,  suc- 
ceeded in  introducing  four,  and  only  four,  methyl  radicals.  From 
this,  however,  it  does  not  follow  that  putrescin  is  not  a  primary  amin, 
since  cadaverin,  an  unquestioned  primary  diamin,  yields  a  substitu- 
tion compound  containing  only  two  methyl  groups  (see  p.  274). 

The  tetra-methyl  substitution  product  of  putrescin,  C4Hg(CIl3)^N2, 
can  be  distilled  without  decomposition.  The  free  base  crystallizes 
in  long  prisms.  The  hydrochlorid  forms  small  needles  which  are 
easily  soluble  ;  with  phosphotungstic  acid  it  gives  a  white  crystalline 
precipitate,  with  phosphomolybdic  acid  a  yellow  crystalline  precip- 
itate,  with  picric  acid  needles.     Potassium  bismuth  iodid  gives  a 


270  CHEMISTRY  OF  THE  PTOMAINS. 

brownish -red  amorphous  deposit,  while  the  potassium  mercuric  iodid 
forms  prisms.  Gold  chlorid  yields  difficultly,  and  platinum  chlorid 
easily  soluble  octahedra ;  aqueous  mercuric  chlorid  forms  needles. 
The  aurochlorid  has  the  formula  OgHggNg.AuCl^. 

This  tetra-methyl  derivative  of  putrescin  is  enormously  poisonous 
as  compared  with  putrescin.  The  symptoms  are  the  same  as  those 
produced  by  muscarin  or  neurin.  They  are  :  abundant  salivation  ; 
dyspnoea — respiration  at  first  increases,  then  decreases  ;  contraction  of 
the  pupils  ;  paralysis  of  the  muscles  of  the  limbs  and  trunk  ;  increased 
peristaltic  action  of  the  intestines,  ejaculation  ot  semen,  dribbling  of 
urine,  and  finally  violent  clonic  convulsions.  In  the  case  of  mice 
and  guinea-pigs  the  convulsions  are  prominent  immediately  after  the 
injection  of  the  poison. 

Putrescin  hydrochlorid,  C^Hj2N2.2HCl,  forms  long  colorless  need- 
les, which  are  very  easily  soluble  in  water ;  difficultly  so  in  dilute 
alcohol ;  entirely  insoluble  in  absolute  alcohol,  and  can  thus  be  sep- 
arated from  cadaverin  hydrochlorid.  To  accomplish  this  separation 
it  is,  perhaps,  better  to  dissolve  the  mixture  of  the  hydrochlorids  in 
hot  96  per  cent,  alcohol.  On  cooling  the  solution  thus  obtained  the 
putrescin  salt  crystallizes  out,  whereas  that  of  cadaverin  remains  in 
solution.  Putrescin  hydrochlorid  differs  from  cadaverin  hydro- 
chlorid in  that  it  is  not  hygroscopic  and  can  be  exposed  for  days  to 
the  air  without  showing  any  change  on  the  surface  of  the  crystals. 

For  the  behavior  of  the  free  base  and  the  hydrochlorid  to  alka- 
loidal  reagents  see  Table  I.  Putrescin  is  not  toxic,  though  it  pos- 
sesses some  marked  physiological  properties  (see  cadaverin,  page  273). 
According  to  Scheurlen,  putrescin,  like  cadaverin,  produces  inflam- 
mation, suppuration  and  necrosis.  It  is  not  poisonous  to  dogs 
(Udranszky  and  Baumann).     It  is  optically  inactive. 

The  platinochlorid,  C,Hj2N2.2HCl.PtCl,  (Pt=  39.16  per  cent.), 
often  appears  under  the  microscope  in  the  form  of  cholesterin-like 
plates.  In  the  pure  condition  it  appears  as  six-sided  plates,  which  are 
superposed  in  layers.  The  crystals  possess  a  splendid  silvery  luster, 
and  are  rather  difficultly  soluble  in  cold  water ;  less  so  in  hot  water. 

The  aurochlorid,  C,Hj2N2.2HC1.2AuCl3  +  2H2O,  crystallizes  like- 
wise in  plates,  which  are  difficultly  soluble  in  cold  water.  It 
can,  therefore,  be  readily  separated  from  cadaverin  aurochlorid, 
which  is  easily  soluble  in  water.  The  water  of  crystallization  can 
be  driven  off  completely  only  at  110°  (Brieger).  According  to 
Bocklisch,  it  loses  this  water  on  standing  over  sulphuric  acid,  or  on 
heating  at  100°. 

The  picrate,  QJ1^^^.1C^Yi.ll^0^)pB.,  is  difficultly  soluble,  and 
crystallizes  from  a  hot  aqueous  solution  in  needles  ;  from  hot  aque- 
ous alcohol,  on  cooling,  in  yellow  plates.  It  begins  to  brown  at 
230°,  and  on  further  heating  becomes  darker,  till  finally,  at  250°,  it 
decomposes  with  rapid  evolution  of  gas  (Bocklisch). 


CADAVERIN.  271 

The  carbonate  is  crystalline. 

The  mercury  double  salt  is  easily  soluble  in  a  large  quantity  of 
water,  and  can  thus  be  separated  from  the  cadaverin  salt,  which  is 
difficultly  soluble.  From  hot  concentrated  aqueous  solution  it  crys- 
tallizes in  needles. 

The  dibenzoyl-putrescin,  C^Hg(NHCOCgH5)2,  forms  silky  plates 
or  long  needles,  which  are  more  difficultly  soluble  in  hot  alcohol  than 
those  of  the  cadaverin  compound.  From  this  solution  it  is  repre- 
cipitated  by  addition  of  water  or  ether.  Its  melting-point  is  175°. 
It  sublimes  without  decomposition. 

Cadaverin,  C5lIj^N2,  is  a  diamin  isomeric  with  saprin  and  neu- 
ridin,  and,  like  the  latter,  it  occurs  very  frequently  in  decomposing 
animal  tissues.  Twelve  isomers  of  this  composition  are  possible. 
Another  isomer,  gerontin  (see  next  chapter),  has  been  described  by 
Grandis  (1890).  It  is  a  very  striking  fact  that  in  ordinary  putre- 
faction as  cholin  disappears  the  diamins  appear  and  increase  in 
quantity  according  as  the  time  of  putrefaction  is  extended.  It  is 
also  worthy  of  note  that  cadaverin  appears  in  putrefaction  before 
putrescin.  It  has  been  obtained  by  Brieger  (1885)  from  human 
lungs,  hearts,  livers,  etc.  (hence  the  name),  which  were  allowed  to 
putrefy  at  the  ordinary  temperature  for  three  days ;  from  the  same 
organs,  and  from  horseflesh,  after  four  months  in  a  closed  vessel  at 
—  9°  to  -I-  5°  ;  from  horseflesh  after  four  months  at  15°,  together 
with  cholin,  and  probably  muscarin  (Gulewitsch)  ;  from  putrid  mus- 
sel after  sixteen  days ;  from  putrid  egg  and  blood-albumin.  It 
seems  to  be  a  constant  product  of  the  growth  of  the  comma  bacillus, 
irrespective  of  the  soil  on  which  it  is  cultivated. 

Bocklisch  has  isolated  it  from  perch  and  pike,  six  days  in  mid- 
summer ;  from  herring,  twelve  days  in  spring  ;  from  haddock,  two 
mouths  at  a  low  temperature ;  from  cultivations  of  Finkler  and  Prior's 
vibrio  proteus  on  beef-broth,  thirty  to  thirty-five  days  at  37°  to  38° 
{Ber.  chem.  Ges.,  20,  1441).  Cadaverin  seems  to  be  a  constant  prod- 
uct of  the  activity  of  the  genus  vibrio,  and  it  does  not  usually  occur 
in  cultures  in  which  this  genus  is  absent.  Thus,  it  is  not  present  in 
the  excrements  of  healthy  or  typhoid  patients,  although  Jakowski  did 
find  it  in  the  intestinal  contents  in  a  case  of  fistula.  It  has  not  been 
obtained  from  cultures  of  Emmerich's  bacillus,  of  Eberth's  bacillus, 
and  of  the  pyogenic  bacteria.  It  occurs  in  cultures  of  the  bacillus 
of  hog  cholera  (Schweinitz)  ;  and  of  the  bacillus  piscicidus  agilis 
(Sieber).  Oechsner  de  Coninck  has  found  it  in  putrid  jelly-fish 
(Hugounenq).  It  is  present  with  putrescin  in  the  urine  and  feces  of 
cystinuria  (Udranszky  and  Baumann  (1888),  see  page  265).  The 
odor  of  cholera  stools  and  the  breath  of  cholera  patients  may  be  pos- 
sibly due  to  cadaverin,  although  Roos  has  not  been  able  to  obtain 
diamins  from  the  rice-water  discharges  of  cholera.     In  one  of  four 


272  CHEMISTRY  OF  THE  PTOMAINS. 

cases  a  small  aniouut  of  a  dibenzoyl-compound,  crystallizing  in  small 
white  plates  and  needles,  and  melting  at  175°-177°,  was  obtained. 
This  corresponds  with  the  putrescin  compound.  No  diamins  were 
found  in  two  cholera-urines.  It  would,  therefore,  seem  that  in 
Asiatic  cholera  diamins  are  not  usually  found  in  the  feces,  and  since 
they  are  present  in  the  feces  and  urine  of  cystinuria  without  bad  re- 
sults, it  is  evident  that  they  cannot  exercise  any  great  action  as  in- 
testinal poisons  in  cholera. 

In  a  diarrhoea,  where  a  coliform  bacillus  was  present,  Roos  found 
both  cadaverin  and  putrescin  in  the  discharges,  but  not  in  the  urine 
of  one  case.  In  another  case  cadaverin  was  likewise  probably 
present.  Werigo  has  reported  cadaverin  from  the  intestinal  con- 
tents of  a  woman  with  intestinal  fistula.  He  would  consider  cadav- 
erin as  a  normal  product  of  pancreatic  digestion. 

Brieger  was  the  first  to  show  that  diamins  were  absent  from 
normal  feces.  Baumann  and  Udranszky  confirmed  this  observation 
with  reference  to  man  and  the  dog.  The  discharges  of  various 
diseases  gave  negative  results  except  in  typhoid  stools,  where  a 
very  small  amount  of  di benzoyl  compounds,  melting  at  140°,  was 
found.  Roos,  in  1891,  was  able  to  find  but  two  cases  with  diamins 
in  the  feces.  In  one  case  of  dysentery  and  malaria  of  tropical  origin 
cadaverin  was  found,  and  in  a  case  of  cholerine  putrescin  was  de- 
tected. It  has  also  been  obtained  from  caviar.  Lobisch  and  Roki- 
tansky  have  reported  it  in  bronchiectasic  sputum.  Werigo  has 
obtained  it  from  pancreas  extracts  before  putrefaction  has  set  in, 
while  Garcia  isolated  it  from  putrefying  meat  and  pancreas  together 
with  putrescin  and  hexamethylenediamin. 

At  least  one  definite  source  of  cadaverin  is  known  and  that  is 
the  hexon  base  lysin  (which  see).  By  allowing  lysin  or  di-amido 
caproic  acid  to  putrefy  Ellinger  (1900)  obtained  cadaverin.  The 
presence  of  the  lysin  group  in  the  proteid  molecule  is  therefore  nec- 
essary to  the  formation  of  this  diamin.  Thus,  while  lysin  forms  in 
sterile  auto-digestion  of  the  pancreas,  in  putrefaction  of  the  latter 
cadaverin  results. 

Cadaverin  occurs  in  the  mercuric  chlorid  precipitate,  from  which 
it  is  isolated  according  to  the  methods  given  on  pages  266  and  278. 
For  its  isolation  and  separation  from  putrescin  by  the  use  of  benzoyl 
chlorid,  see  page  266. 

This  base  was  at  first  ascribed  the  formula  CglljgNg,  but  subsequent 
researches  led  Brieger  and  Bocklisch  to  the  adoption  of  the  formula 
CgHj^N^.  In  1883,  Ladenburg  prepared,  as  the  first  step  in  the  syn- 
thesis of  piperidin,  a  base,  pentamethylenediamin,  possessing  the  same 
empirical  formula  as  cadaverin,  and  later  (jBer.  chem.  Ges.,  18,  2956) 
he  showed  the  possibility  of  the  identity  of  these  two  bases.  This  led 
to  their  direct  comparison  and  the  successful  establishment  of  their 
identity.     In  fact,  Ladenburg,  as  a  crucial  test  of  the  identity,  con- 


CADAVERIN.  273 

verted  cadaverin  into  piperidin,  and  found  the  latter  base  to  agree 
entirely  in  its  chemical  and  physical  properties  with  those  of  the 
natural  alkaloid  {Bei\,  19,  2586).  Ladenburg,  however,  observed 
one  apparent  difference  between  cadaverin  and  pentamethylenedi- 
amiu,  and  that  was  in  the  composition  of  the  mercury  double  salts. 
That  of  the  former  base,  whether  obtained  from  alcoholic  or  aqueous 
solution  (Bocklisch,  Ber.,  20,  1441),  was  found  to  combine  with  four 
molecules  of  mercuric  chlorid;  whereas  the  double  salt  of  penta- 
methylenediamin  was  found  by  Ladenburg  to  contain  only  three 
molecules  of  mercuric  chlorid.  Subsequently  he  found  that  he  had 
prepared  this  salt  by  mixing  the  aqueous  solutions  of  the  hydro- 
chlorid  of  the  base  and  of  the  mercuric  chlorid  in  the  molecular  ratio 
of  1  to  4,  and  on  using  a  larger  excess  of  mercuric  chlorid  he  ob- 
tained a  salt  containing  four  molecules  of  mercuric  chlorid  {Ber.,  20, 
2216).  The  complete  identity  of  these  two  bases  has,  therefore,  been 
established.     The  constitutional  formula  of  cadaverin  is,  therefore  : 

NHj  —  CHj  —  CH2  —  CHj  —  CHj  —  CK,  —  NHj  • 

Cadaverin  can  be  prepared  synthetically  according  to  Ladenburg's 
method.  For  this  purpose  trimethylene  bromid  is  converted  into 
the  cyanid,  and  this  is  then  reduced  by  sodium  in  absolute  alcohol. 

Cadaverin  forms  a  somewhat  thick,  water-clear,  syrupy  liquid, 
which  possesses  an  exceedingly  unpleasant  odor,  resembling  some- 
what that  of  coniin  (piperidin)  and  of  semen.  When  dehydrated 
with  potassium  hydrate  it  boils  at  115°-120°  (Brieger).  It  boils 
at  175°  (Brieger,  III.,  98),  and  fumes  in  the  air.  The  base  eagerly 
absorbs  carbonic  acid  from  the  air,  and  solidifies  into  a  crystalline 
mass,  the  carbonate.  It  is  volatile  with  steam,  and  can  be  distilled, 
without  decomposition,  even  in  presence  of  sodium  or  barium  hydrate 
or  soda-lime.  Neuridin,  its  isomer,  decomposes  under  these  circum- 
stances. When  heated  with  alcoholic  potash  and  chloroform  it  does 
not  give  the  isonitril  reaction,  nor  does  it  give  the  characteristic  odor 
of  oil  of  mustard  on  treatment  with  carbon  disulphid  and  mercuric 
chlorid.  The  absence  of  these  reactions  at  first  induced  Brieger  to 
conclude  that  cadaverin  and  putrescin  were  not  primary  amins,  but 
Ladenburg  (1885)  showed  that  this  conclusion  was  not  justifiable. 
These  two  reactions  are  given  by  primary  monamins,  but  in  this  case 
they  are  not  given  by  cadaverin,  a  primary  diamin.  It  is  probable 
that  this  behavior  holds  true  for  all  diamins. 

Cadaverin  is,  undoubtedly,  identical  with  the  so-called  "  animal 
coniin,"  which  has  been  isolated  at  various  times  from  cadavers. 

Cadaverin  and  putrescin  were  at  first  regarded  as  physiologically 
indifferent,  but  more  recent  investigations  by  Scheurlen,  Grawitz, 
and  others,  show  that  both  these  bases  are  capable  of  producing  strong 
inflammation  and  necrosis.  According  to  Behring,  in  large  doses  it 
is  poisonous  to  mice,  rabbits,  and  guinea-pigs ;  it  is  not  poisonous  to 
18 


274  CHEMISTRY  OF  THE  PTOMAINS. 

dogs  (Udr^nszky  and  Baumann).  The  synthetic  putrescin  accord- 
ing to  Pohl  produces  marked  dyspncea  in  rabbits  ;  larger  doses  (0.4  g.) 
cause  persistent  tonic  convulsions  and  paralyses  of  the  extremities. 
Cadaverin  is  one  of  those  substances  which  can  set  up  suppuration 
in  the  absence  of  bacteria.  In  cholera  Asiatica  the  necrosis  of  the 
intestinal  epithelium  is  quite  common,  and  it  would  seem  that  this 
pathological  change,  as  well  as  the  muscular  spasms  and  algidity, 
are  due  to  the  presence  of  these  bases.  It  should  be  noted,  however, 
that  Udranszky  and  Baumann  failed  to  obtain  any  sign  of  intestinal 
irritation  on  feeding  dogs  enormous  doses  of  cadaverin ;  and,  more- 
over, Roos  (page  271)  failed  to  find  these  bases  in  the  feces  of  chol- 
era. Besides  these  local  effects,  they  prevent,  even  in  small  quan- 
tity, the  coagulation  of  blood,  and  render  it  "laky."  According  to 
Grawitz,  cadaverin  seems  to  hinder  the  growth  of  bacteria.  The 
cystitis  observed  in  cystinuria  may  possibly  be  due  to  the  presence 
of  cadaverin  and  putrescin  *in  the  urine.  Both  bases  are  optically 
inactive. 

When  cadaverin  is  treated  with  methyl  iodid,  a  base  is  obtained 
the  hydrochlorid  of  which  gives  with  platinum  chlorid  a  double  salt, 
having  the  composition,  C^^^(CJlX'N ^.21IC\.FtC\,.  This  new 
base,  therefore,  is  cadaverin  in  which  two  atoms  of  hydrogen  have 
been  replaced  by  two  methyl  radicals.  The  platinochlorid  of  this 
derivative  forms  long,  clear  red  needles,  which,  unlike  those  of  ca- 
daverin, do  not  change  their  shape  on  repeated  recrystallization.  It 
is  moderately  difficultly  soluble  in  water  (Brieger,  II.,  41).  Since 
cadaverin  is  a  primary  diamin  it  should  combine  with  six  molecules 
of  methyl  iodid  to  form  a  saturated  compound.  This,  however,  has 
not  been  obtained. 

The  hydrochlorid,  C5lIj^]Sr2.2HCl,  crystallizes  in  beautiful,  long 
deliquescent  needles  (Brieger).  According  to  Bocklisch  it  forms 
long,  colorless  needles  or  prisms ;  crystallizes  from  alcohol  in  plates, 
and  is  not  deliquescent  except  on  long  standing.  From  95  per 
cent,  alcohol  it  crystallizes  in  short,  pointed  stellate  prisms,  which 
are  not  deliquescent  (Gulewitsch).  On  evaporation  of  an  aqueous 
solution  it  forms  very  long  prismatic  crystals.  It  shows  no  circum- 
polarization.  It  possesses  a  slight  bitter  taste  (Gulewitsch).  It  is 
soluble  in  water,  alcohol,  alcohol-ether ;  but  is  insoluble  in  absolute 
alcohol,  ether,  etc.  It  can  readily  be  separated  from  putrescin  hy- 
drochlorid by  its  solubility  in  96  per  cent,  alcohol  (Bocklisch). 
The  strictly  pure  base,  as  well  as  the  hydrochlorid,  does  not  give  a 
blue  color  with  ferric  chlorid  and  potassium  ferricyanid.  For  reac- 
tions of  the  hydrochlorid  and  of  the  free  base,  see  Table  I. 

Cadaverin  hydrochlorid  on  dry  distillation  decomposes  into  NHj, 
HCl,  and  piperidin,  C^HuN.  The  latter  is  a  well-known  poisonous 
alkaloid  which  exists  in  the  combined  state  in  black  pepper.  It  is 
not  known  whether  this  change,  whereby  the  non-poisonous  cadav- 


CADAVERIN.  275 

erin  is  converted  into  a  toxic  base,  can  take  place  under  the  influence 
of  bacteria  during  the  process  of  putrefaction,  or  not.  However,  it 
does  not  seem  improbable  that  this  simple  chemical  change  should 
be  effected  through  the  action  of  living  organisms ;  for  Schmidt  has 
already  shown  that  the  almost  physiologically  indiflFerent  cholin, 
when  subjected  to  the  action  of  the  bacteria  of  hay-infusion,  decom- 
poses into  a  neurin-like  base  possessing  a  muscarin-like  action,  and 
under  certain  conditions  it  yields  a  base  which  in  its  action  resembles 
pilocarpin. 

The  sulphate  likewise  forms  beautiful,  well-formed  needles,  and  in 
its  solubility  corresponds  to  the  hydrochlorid. 

The  platinochlorid,  C5Hj,N2.2HCl.PtCl,(Pt  =  38.08  per  cent.), 
crystallizes  after  some  time,  on  the  addition  of  platinum  chlorid,  to 
a  not  too  concentrated  solution  of  the  hydrochlorid,  in  the  form  of 
long,  beautiful  orange-red  needles  (Bocklisch).  Ordinarily  it  is  ob- 
tained at  first  in  long,  dirty-red  needles,  which  on  repeated  recrys- 
tallization  become  clearer  and  assume  a  form  similar  to  that  of 
ammonium  platinochlorid.  It  forms  chrome-yellow  rhombic  prisms 
which  are  short  and  octahedral-like.  Variation  in  the  crystalline 
form  is  observed  here  as  in  the  case  of  the  mercury  compounds.  In 
polarized  light  they  are  strongly  double  refracting.  It  is  very 
slightly  soluble  in  cold  water ;  can  be  recrystallized  from  hot  water 
(Bocklisch).  Its  solubility  in  water  at  12°  is  1  to  113-114  ;  at  21° 
it  is  1  to  70.8  (Gulewitsch).  It  is  soluble  in  alcohol.  It  decom- 
poses at  235°-236°.  It  does  not  lose  weight  at  125°-135°  ;  at 
195°  it  begins  to  darken  and  melts  with  decomposition  at  215° 
(Gulewitsch). 

The  aurochlorid,  C5H,,N2.2HC1.2AuCl3(Au  =  50.41  per  cent.), 
crystallizes  partly  in  cubes,  and  partly  in  long  needles  which  at  first 
possess  a  bright  lustre,  but  under  the  desiccator  soon  effloresce  and 
become  opaque.  It  crystallizes  from  water  acidulated  with  hydro- 
chloric acid,  in  plates  or  in  large,  long,  very  pretty  orange-yellow 
flat  prisms.  On  rapid  crystallization  bright  platelets  form  (Gule- 
witsch). The  water  of  crystallization  is  completely  removed  on 
standing  over  sulphuric  acid.  It  is  very  easily  soluble,  and  melts  at 
188°  (Bocklisch);  186°-188°  (Gulewitsch). 

The  picrate,  C5Hj^N2.2CgH2(N02)30H,  forms  yellow  plates  which 
are  difficultly  soluble  in  cold  water.  From  hot  water  it  crystallizes 
in  long  prisms,  which  melt  at  221°  with  decomposition.  When 
crystallized  from  95  per  cent,  alcohol  it  forms  long  yellow  needles, 
which  are  difficultly  soluble  in  cold,  more  easily  in  hot  95  per  cent, 
alcohol.  It  is  insoluble,  or  very  difficultly  so,  in  absolute  alcohol, 
and  can  be  recrystallized  from  hot  dilute  alcohol. 

Cadaverin  hydrochlorid  combines  with  mercuric  chlorid,  when  the 
aqueous  solutions  of  these  two  salts  are  mixed  in  the  molecular  ratio 
of  1  to  4,  to  form  C5H,,N2.2HC1.3HgCl2.     This  salt  can  be  recrys- 


276  CHEMISTRY   OF  THE  PTOMAINS. 

tallized  from  hot  water  (Ladenburg).  "When  an  excess  of  mercuric 
chlorid  is  used  the  double  salt  has  the  composition  C5Hj^N2.2HCl. 
4HgCl2.  This  last  salt  melts  at  216°  (Ladenburg) ;  at  214°  (Bock- 
lisch).  It  is  difficultly  soluble  in  cold  water ;  easily  in  hot  water  at 
21°  (1-32.5,  Gulewitsch) ;  from  hot  water  it  crystallizes  in  needles 
or  plates  (Bocklisch).  On  heating  even  on  the  water-bath  it  loses 
weight.  At  125°-135°  it  loses  18.33  per  cent,  of  its  weight, 
due  to  volatilization  of  mercuric  chlorid  (Gulewitsch).  As  pointed 
out  by  Brieger,  it  is  quite  probable  that  other  mercuric  compounds 
exist  than  those  mentioned.  Gulewitsch  (1894)  showed  inter- 
esting polymorphism  of  the  mercury  salts  of  cadaverin.  He  in- 
clined to  the  belief  that  it  may  form  compounds  with  more  than 
three  or  four  molecules  of  mercuric  chlorid.  On  heating  mercuric 
chlorid  is  given  ofP,  and  hence  the  varieties  in  form.  This  variation 
in  form  is,  therefore,  not  necessarily  due  to  impurities.  When  first 
obtained  the  cadaverin  mercurochlorid  forms  warty  aggregates  of 
dark-brown  prisms  with  pointed  ends.  By  repeated  recrystallization 
from  water  it  eventually  forms  single  or  stellate  rhombic  plates,  and 
on  further  crystallization  very  thin,  elongated,  six-sided,  or  triangu- 
lar plates  result. 

The  neutral  oxalate,  Cgllj^Ng.HgCjO^  -|-  2H2O,  was  prepared  by 
Bocklisch  by  adding  a  little  less  than  the  calculated  quantity  of  al- 
coholic oxalic  acid  to  the  cadaverin.  The  precipitate  may  be  re- 
crystallized  from  hot  dilute  alcohol,  when  it  is  obtained  in  the  form 
of  needles,  which  melt  at  about  160°,  and  at  the  same  time  give 
off  gas. 

The  acid  oxalate,  C^^^^.21Af^f)^  -}-  HgO,  is  made  by  bringing 
the  neutral  salt  into  alcoholic  oxalic  acid.  It  is  soluble  in  hot  dilute 
alcohol,  and  recrystallizes  from  it  in  quadratic  plates,  sometimes  in 
glistening  needles.  It  melts  at  143°  with  decomposition.  After  it 
has  been  dried  over  sulphuric  acid  it  loses,  on  being  heated  to  105°— 
110°,  one  molecule  of  water  (Bocklisch,  Ber.,  20,  1441).  The  in- 
solubility of  these  oxalates  in  absolute  alcohol  shows  the  fallacy  of 
Tamba's  distinction  between  ptomains  and  vegetable  alkaloids. 

The  dibenzoyl-derivative,  CgH^p(NHCOCgH5)2,  crystallizes  in  long 
or  small  needles  and  plates,  readily  soluble  in  alcohol,  difficultly  so 
in  ether,  and  insoluble  in  water ;  hence  the  alcoholic  solution  can 
be  precipitated  by  addition  of  water  or  ether  (separation  from  the 
putrescin  compound,  see  page  266).  It  melts  at  129°-130°  ;  at 
130.5°-131.5°  (Gulewitsch).  It  is  not  changed  by  boiling  with 
dilute  acids  and  alkalis ;  but  boiling  with  concentrated  hydrochloric 
or  sulphuric  acid  for  a  long  time  finally  breaks  it  up. 

Neuridin,  C^Hj^Nj,  was  the  first  diamin  isolated  from  animal  tis- 
sues (Brieger,  1883).  It  is  one  of  the  most  common  products  of 
putrefaction,  and  as  such  has  been  obtained  by  Brieger  from  putrid 


NEURIDIN.  277 

horseflesh,  beef,  human  muscle,  five  to  six  days  ;  from  haddock,  five 
days  in  summer ;  from  cheese,  six  weeks  in  summer ;  from  gelatin, 
ten  days  at  53°;  from  decomposing  human  internal  organs,  three  to 
eleven  days ;  from  cultures  of  the  Eberth  bacillus,  with  my  din. 
Bocklisch  has  obtained  it  from  perch,  six  days  in  summer ;  from 
barbel  after  three  days  in  summer. 

It  has  also  been  obtained  from  fresh  eggs  in  the  preparation  of 
cholin  by  heating  with  baryta  ;  and  also  from  fresh  brain  by  heat- 
ing with  2  per  cent,  hydrochloric  acid  (Brieger,  I.,  57-61). 
Ehrenberg  (1887)  found  it  in  poisonous  sausage  and  obtained  it  by 
growing  a  bacillus  from  this  source  on  liver  and  meat  bouillon. 

Neuridin  is  almost  invariably  accompanied  by  cholin,  and  as  the 
duration  of  putrefaction  increases  the  latter  gradually  decreases  in 
amount  and  yields  a  corresponding  increase  in  trimethylamin, 
whereas  the  yield  of  neuridin  increases  from  day  to  day.  The 
amount  of  neuridin  formed  depends  upon  the  nature  of  the  organ 
employed  in  putrefaction.  The  greatest  yield  is  obtained  from  gel- 
atinous tissues  such  as  intestines  ;  and  especially  from  pure  gelatin. 
On  the  other  hand  such  tissues  as  the  spleen  and  liver  yield  but 
little. 

Neuridin  comes  down  in  the  mercuric  chlorid  precipitate  (some- 
times it  occurs  in  the  filtrate),  and  can  then  be  isolated  from  the 
other  bases  present  in  a  number  of  ways.  One  method  is  given 
under  gadinin.  Another  convenient  method  of  separation  is  to  pre- 
cipitate it  from  alcoholic  solution  by  alcoholic  picric  acid.  The 
picrate  thus  obtained  is,  for  the  purpose  of  further  purification,  re- 
crystallized  from  absolute  alcohol,  then  decomposed  by  extracting  its 
acid  solution  with  ether  (to  remove  the  picric  acid)  and  evaporating 
the  aqueous  solution  to  dryness.  The  residue  is  now  extracted  with 
alcohol  and  the  alcoholic  solution  precipitated  by  alcoholic  platinum 
chlorid.  The  platinochlorid  can  now  be  recrystallized  from  hot 
water. 

The  free  base,  as  obtained  by  the  treatment  of  the  hydrochlorid 
with  moist,  freshly  precipitated  silver  oxid,  possesses  an  extremely 
repulsive  odor,  similar  to  that  of  human  semen. 

On  evaporation  of  its  aqueous  solution  it  yields  a  gelatinous-like 
mass,  and  at  the  same  time  slowly  decomposes.  It  does  not  crys- 
tallize when  evaporated  in  a  vacuum,  and  decomposes  even  under 
these  conditions.  The  same  disagreeable  odor  is  obtained  when  the 
hydrochlorid  is  warmed  with  potassium  hydrate.  Brieger  (I.,  24) 
regards  this  decomposition  product  of  neuridin  as  an  oxidation 
product  of  the  original  substance. 

The  free  base  is  very  readily  soluble  in  water,  but  is  insoluble  in 
ether  and  absolute  alcohol ;  difficultly  soluble  in  amyl  alcohol.  It 
gives  white  precipitates  with  mercuric  chlorid,  neutral  and  basic  lead 
acetates.     When  distilled  with  fixed  alkali  it  yields  di-  and  tri- 


278  CHEMISTRY  OF  THE  PTOMAINS. 

methylamin,  thus  probably  showing  some  relation  to  neurin,  hence 
the  name  neuridin.  It  does  not  give  Hofmann's  isonitril  reaction, 
but  it  does  not  follow  from  this,  as  shown  under  cadaverin,  that  it 
may  not  be  a  primary  diamin.  It  is  isomeric  with  cadaverin, 
saprin,  and  gerontin. 

The  hydrochlorid,  C5lIj^N2.2IICl,  crystallizes  in  long  needles 
which  are  extremely  soluble  in  water  and  in  dilute  alcohol,  but 
are  insoluble  in  absolute  alcohol,  ether,  chloroform,  petroleum 
ether,  benzene,  amyl  alcohol,  etc.  Its  insolubility  in  absolute  alco- 
hol may  be  used  to  effect  a  separation  from  cholin  hydrochlorid. 
It  can  be  recrystallized  from  slightly  warm  dilute  alcohol.  Al- 
though the  pure  salt  is  insoluble  in  the  reagents  just  given,  never- 
theless, in  the  presence  of  other  animal  matter,  it  is  dissolved  in 
greater  or  less  quantity,  and  hence  can  be  obtained  by  the  Stas-Otto 
as  well  as  by  the  Dragendorff  method.  The  crystals  resemble  urea 
in  form.  On  heating  very  cautiously  the  salt  sublimes,  and  at  the 
same  time  appears  to  undergo  a  partial  internal  decomposition,  inas- 
much as  many  of  the  groups  of  needles  in  the  sublimate  are  colored 
red  or  blue.  For  the  behavior  of  the  hydrochlorid  with  the  alkaloidal 
reagents,  see  Table  I. 

Pure  neuridin  is  not  poisonous,  but  as  long  as  it  is  contaminated 
with  other  putrefaction  products  it  possesses  a  toxic  action  similar  to 
that  of  peptotoxin.  This  holds  true  for  the  other  non-poisonous 
bases. 

The  platinochlorid,  CgHj^N2.2IICl.PtCl4,  crystallizes  in  beautiful 
flat  needles.  Recrystallized  from  hot  water,  it  forms  aggregations 
of  small,  clear,  yellow  needles.  It  is  readily  soluble  in  water,  from 
which  it  is  precipitated  on  the  addition  of  alcohol. 

The  aurochlorid,  0^11^^1^^.211012^:101^,^  is  rather  difficultly  sol- 
uble in  cold  water  (Bocklisch),  and  crystallizes  on  cooling  of  the  hot, 
saturated  solution  in  bunches  of  clear,  yellow,  short  needles. 

The  picrate,  CgH^^N2.2CgIl2(N02)30H,  can  be  recrystallized  from 
boiling  water,  in  which  it  is  very  difficultly  soluble,  in  the  form  ot 
needles  united  in  plumose  groups.  It  is  almost  insoluble  in  cold 
water ;  less  difficultly  soluble  in  alcohol.  It  is  not  fusible,  but  be- 
gins to  brown  and  give  off  yellow  vapors  at  230°,  and  carbonizes 
completely  at  250°. 

Saprin,  CgHj^N2,  was  found  in  human  livers  and  spleens  after 
three  weeks  putrefaction  (Brieger,  II.,  30,  46,  58).  It  occurs  to- 
gether with  cadaverin,  putrescin,  and  mydalein  in  the  mercuric 
chlorid  precipitate.  To  separate  these  bases  Brieger  (1885)  used  the 
following  process  :  The  mercury  salts  were  decomposed  with  hydro- 
gen sulphid,  the  filtrate  evaporated  to  dryness,  and  the  residue  then 
extracted  with  alcohol.  The  putrescin  hydrochlorid  is  insoluble  in 
alcohol,  and   is  thus  removed.     The  alcoholic  solution  was  treated 


HEXAMETHYLENEDIA  MIX.  279 

with  platinum  chlorid,  which  precipitated  the  greater  part  of  the 
cadaverin.  The  mother-liquor,  on  concentration,  yielded  a  mixture 
of  the  platinochlorids  of  cadaverin  and  saprin.  Each  successive 
crop  contained  more  of  the  saprin  double  salt.  The  two  kinds  of 
crystals  were  now  separated  by  means  of  a  magnifying  glass.  The 
saprin  platinochlorid  thus  obtained  was  finally  purified  by  repeated 
recrystallization  from  water.  The  mother-liquor,  after  the  removal 
of  the  saprin  platinochlorid,  contains  the  mydalein  salt,  which  on  ac- 
count of  its  solubility  in  water,  crystallizes  only  on  concentration,  or 
on  standing  under  a  desiccator.  The  mercuric  chlorid  filtrate  con- 
tains some  mydalein  and  the  ptomain,  which  yields  a  platinochlorid 
containing  28.40  per  cent,  platinum. 

The  free  base  is  a  diamin,  and  was  first  ascribed  the  formula 
CjHjgNg.  It  appears,  however,  to  be  isomeric  with  cadaverin  and 
neuridin.  The  term  saprin  is  derived  from  the  Greek  aazpo^,  signi- 
fying putrid.  It  possesses  a  weak  pyridin-like  odor,  and  can  be  dis- 
tilled with  steam  or  with  potassium  hydrate  without  undergoing 
decomposition.  In  its  reactions  it  behaves  the  same  as  cadaverin, 
except  that  it  gives  an  amorphous  precipitate  with  potassium  bis- 
muth iodid,  whereas  cadaverin  gives  a  crystalline  precipitate.  The 
free  base  gives  an  immediate  intense  blue  color  with  ferric  chlorid 
and  potassium  ferricyanid. 

The  hydrochlorid,  C.Hj^N2.2IICl,  forms  flat  needles  which  are  not 
hygroscopic  (distinction  from  cadaverin  hydrochlorid).  Its  reactions 
are  the  same  as  those  of  cadaverin  hydrochlorid.  (See  Table  I.)  It 
is,  however,  tinged  slightly  blue  by  a  mixture  of  ferric  chlorid  and 
potassium  ferricyanid,  whereas  the  free  base  gives  an  intense  blue. 
It  differs  from  cadaverin  in  that  it  does  not  give  the  reddish-brown 
color  with  potassium  bichromate  and  sulphuric  acid.  Again,  it  forms 
no  aurochlorid  ;  while,  on  the  other  hand,  cadaverin  hydrochlorid 
yields  an  easily  soluble  salt,  crystallizing  in  splendid  needles. 

The  platinochlorid,  C5H^^N2.2HCl.PtCl^,  forms  parallel,  aggre- 
gated, pointed  crystals,  which  are  somewhat  soluble  in  water,  and 
are  thus  distinguished  from  cadaverin  platinochlorid,  which  crystal- 
lizes in  rhombs,  and  is  difficultly  soluble  in  water. 

Physiologically,  it  is  indifferent. 

Hexamethylenediamin,  CgH^gN,. — This  compound  was  found  by 
Garcia  ifi  decomposing  meat  and  pancreas  mixture,  seven  days  at 
30°,  by  the  benzoyl  chlorid  method,  together  with  cadaverin  and 
putrescin.  The  dibenzoyl-putrescin  is  removed  in  the  usual  way 
by  precipitating  the  alcoholic  solution  of  the  mixed  benzoyl  diamins 
with  ether.  The  separation  of  cadaverin  from  the  new  compound  is 
more  difficult  owing  to  the  great  similarity  in  the  solubilities  of  the 
two  compounds.  Garcia  succeeded  in  effecting  a  separation  by  dis- 
solving the  mixture  in  alcohol,  and  raising  the  temperature  on  a 


280  CHEMISTRY  OF  THE  PT0MAIN8. 

water-bath  to  70°  ;  by  gradual  addition  of  water  (50  volumes), 
taking  care  not  to  allow  the  temperature  to  rise  above  70°,  at  which 
temperature  the  solution  is  left  for  twenty  or  thirty  minutes ;  may 
be  raised  to  90°  and  kept  there  for  one  hour.  The  solution  is  then 
rapidly  filtered  through  an  asbestos  plug  with  the  aid  of  a  pump. 
On  cooling,  bright,  long  crystalline  plates  and  needles  separate  from 
the  filtrate.  The  residue,  dissolved  in  alcohol  and  again  treated  by 
this  process,  finally  yields  a  residue  of  pure  benzoyl-cadaverin. 
The  crystals  formed  in  the  filtrate  melt  at  124.5°-125°  ;  cadaverin 
compound  at  129°-130°. 

The  dibenzoyl  compound  on  heating  with  equal  parts  of  alcohol 
and  concentrated  hydrochloric  acid  on  a  water-bath  for  forty-eight 
hours  is  decomposed.     The  hydrochlorid  is  not  deliquescent. 

The  platinochlorid,  CgH.gN^-^HCl.PtCl,  (Pt  =  37.01),  crystallizes 
from  hot  water  on  cooling  in  elongated,  well -formed  needles 
(rhombic  system)  of  dark -orange  color.  Some  were  more  than  1 
cm.  long.  The  crystallographic  characters  of  the  salt  agree  in  all 
respects  with  those  of  the  cadaverin  compound  with  which  it  is 
therefore  isomorphous.  It  is  easily  soluble  in  water,  difficultly  in 
strong  alcohol. 

Gold  chlorid  produces  no  precipitate  in  aqueous  or  alcoholic  solu- 
tion of  the  base.  Picric  acid  gives  a  compound  easily  soluble  in 
water  and  in  absolute  alcohol.  It  crystallizes  in  needles  and  plates  ; 
at  200°  becomes  brown,  and  at  210°  it  decomposes.  In  its  be- 
havior to  gold  chlorid  it  resembles  saprin. 

This  base  is  not  present  in  cystinuria. 

A  Base,  C^Hj^Ng. — Until  very  recently  the  nature  of  the  basic 
substances  which  are  formed  as  products  of  the  alcoholic  fermenta- 
tion of  sugar  or  molasses  has  been  but  little  understood.  Kramer 
and  Pinner,  in  1869,  found  in  crude  fusel  oil  a  small  quantity  of  a 
volatile  base  which  they  apparently  identified  with  a  coUidin.  This 
observation  was  confirmed  by  Ordonneau  and  others;  and  in  1888 
Morin  contributed  an  elaborate  paper  upon  the  bases  formed  during 
alcoholic  fermentation.  The  portion  of  crude  fusel  oil  which  boils 
above  130.5°  was  extracted  with  slightly  acidulated  water,  the  acid 
aqueous  solution  thus  obtained  was  made  alkaline,  and  the  oily  bases 
which  were  thus  set  free  were  then  distilled  with  vapor  of  water. 
The  free  bases  were  then  dried  over  potassium  hydrate  and  sub- 
jected to  fractional  distillation.  Three  fractions  were  thus  obtained, 
boiling  respectively  at  155°-160°,  171°-172°,  and  185°-190°. 
Only  the  second  fraction,  which  boils  at  171°-172°,  was  studied, 
and  was  found  to  possess  the  formula  C^^HjgNj.  Heated  with  con- 
centrated hydrochloric  acid,  it  is  decomposed  in  part  with  the  forma- 
tion of  ammonia.  It  combines  with  ethyl  iodid  to  form  a  yellow 
crystalline  compound  which  is  soluble  in  water  and  alcohol,  insoluble 


SUSOTOXIN.  281 

in  ether.  The  hydrochlorid  crystallizes  in  fine  white  needles,  soluble 
in  water  and  alcohol,  and  but  very  slightly  soluble  in  absolute  ether. 
The  free  base,  as  stated  above,  boils  at  171°-172°,  is  very  soluble 
in  water,  alcohol,  ether,  etc.  When  pure  it  forms  a  colorless, 
strongly  refracting,  very  mobile  oil,  which  possesses  a  characteristic, 
nauseating  odor,  but  slightly  resembling  that  of  the  pyridin  bases. 
Its  density  at  12°  is  0.9826  ;  toward  litmus  paper  the  base  shows 
no  decided  reaction.  The  platinochlorid  is  crystalline  and  is  very 
soluble  in  water  and  alcohol,  slightly  soluble  in  ether.  Potassic 
mercuric  iodid  does  not  precipitate  the  aqueous  solution  of  the  free 
base,  but  in  solutions  of  the  hydrochlorid  it  gives  a  yellow  flocculent 
precipitate,  which  soon  crystallizes  in  long  brilliant  yellow  needles. 
This  reaction  takes  place  readily  in  solutions  of  1  to  1,000,  and  only 
after  some  hours  in  solutions  of  1  to  10,000;  and  is  not  given  by 
the  bases  of  the  pyridic  and  quinolinic  series.  Mercuric  chlorid 
produces  an  immediate  flocculent  precipitate  in  solutions  of  the  base 
having  a  concentration  of  1  to  1,000,  but  requires  some  time  to  ap- 
pear in  1  to  10,000.  Phosphotungstic  acid  gives  an  immediate  white 
precipitate  even  in  a  dilution  of  1  to  1 0,000.  Phosphomolybdic  acid 
in  solutions  of  the  same  strength  yields  a  yellow  precipitate. 

The  physiological  action  of  this  base  has  been  examined  by  R. 
Wurtz  who  found  the  lethal  dose  for  rabbits,  etc.,  to  be  about  one 
gram  per  kilogram  of  body-weight.  It  produces  stupor  and  paralysis 
which  at  first  appears  in  the  rear  extremities  ;  the  sensibility  becomes 
diminished  and  the  pupils  are  dilated  and  unresponsive  to  light ;  the 
rate  of  heart-beat  is  lowered,  and  the  rectal  temperature  falls  as  low 
as  35°  ;  death  follows  a  more  or  less  prolonged  coma. 

Tanret  obtained  by  the  action  of  ammonia  on  glucose  a  number  of 
bases,  to  which  he  applied  the  generic  name  of  glucosins.  One  of 
these,  having  the  formula  Cj^Hj^Ng  (C  =  6),  corresponds  in  its  for- 
mula and  its  general  properties  to  Morin's  base,  C^Hj^Ng  (C  =  12), 
and,  in  fact,  the  two  bases  are  considered  by  Tanret  to  be  identical. 

It  is  interesting  to  note  in  this  connection  that  alkaloidal  bases  have 
been  found  in  petroleum  by  Bandrowski,  and  that  similar  basic  sub- 
stances have  been  detected  by  Weller  in  paraffin  oil. 

Most  of  the  solvents  in  common  use,  such  as  alcohol,  ether,  chlor- 
oform, benzol,  petroleum  ether,  amyl  alcohol,  etc.,  have  been  shown 
at  different  times  to  contain  basic  pyridin  compounds,  though  ordi- 
narily in  very  minute  quantity.  On  the  other  hand,  Haitinger  has 
found  in  some  specimens  of  amyl  alcohol  as  much  as  0.5  per  cent, 
of  pyridin. 

Susotoxin,  Ci(,H2gN2(?),  is  a  base  isolated  by  Novy  in  1890  from 
cultures  of  the  hog  cholera  bacillus  of  Salmon  (swine  plague  of  Bill- 
ings). It  is  probably  identical  with  the  base  obtained  by  v.  Schwein- 
itz  from  the  same  germ,  although  the  formula  ascribed  to  it  by  him 


282  CHEMISTRY  OF  THE  PTOMAINS. 

* 

is  Cj^HgjNg.  The  free  base  has  not  been  obtained.  The  hydrochlorid 
forms  a  light-yellow  syrup  which  shows  no  tendency  to  crystallize. 
It  is  soluble  in  water  and  in  absolute  alcohol,  and  is  somewhat  hy- 
groscopic. 

When  heated  with  fixed  alkali  it  gives  off  a  strong  amin  odor, 
such  as  is  perceived  on  evaporating  the  original  culture  fluid,  if  it 
happens  to  be  alkaline  in  reaction. 

The  platinochlorid  is  obtained  by  precipitation  as  a  light,  flesh- 
colored,  granular  precipitate.  It  is  readily  soluble  in  water,  from 
which  it  can  be  reprecipitated  by  addition  of  absolute  alcohol. 
From  aqueous  solution,  when  allowed  to  evaporate  slowly,  it  crys- 
tallizes in  long,  thick  needles. 

The  mercurochlorid  is  thrown"  down  from  solutions  of  the  hydro- 
chlorid in  absolute  alcohol,  by  alcoholic  mercuric  chlorid,  as  a 
heavy,  white,  granular  precipitate.  This  readily  dissolves  on  the 
addition  of  a  small  quantity  of  water,  and  can  be  perfectly  reprecip- 
itated by  addition  of  absolute  alcohol.  On  treatment  with  hydrogen 
sulphid  it  is  readily  decomposed,  yielding  the  pure  hydrochlorid. 

The  aurochlorid  is  very  soluble  in  water  and  alcohol.  From  the 
alcoholic  solution  it  may  be  partially  precipitated  by  ether  as  a  light- 
yellow,  oily  precipitate,  which  is  adherent  to  the  sides  and  bottom  of 
the  tube. 

The  base  is  toxic  only  in  relatively  large  doses,  as  seen  from  the 
following  experiment.  About  100  milligrams,  dissolved  in  a  little 
water,  were  injected  subcutaneously  into  a  young  rat.  The  animal 
was  at  first  quiet,  apparently  unwilling  to  move.  After  some  inef- 
fectual attempts  at  jumping  it  settled  down  to  a  recumbent  position, 
and  when  placed  on  its  side  was  unable  to  rise.  Respiration  was  at 
first  retarded,  later  increased,  but  toward  the  end  was  again  very 
slow.  Convulsive  tremors  shook  the  body  at  frequent  intervals. 
The  animal  kicked  vigorously.  Reflexes  were  present  almost  to  the 
end.  As  death  approached  the  red  eyes  whitened  and  took  on  a 
glazed,  opaque  appearance.  Death  resulted  in  one  and  a  half  hours. 
The  animal  was  on  its  side,  the  feet  extended.  Post-mortem  exam- 
ination showed  the  heart  arrested  in  diastole,  lungs  rather  pale, 
stomach  contracted,  serum  in  thoracic  cavity,  subcuta  pale  and 
oedematous.  Repeated  doses  of  smaller  quantities  seem  to  confer  a 
partial  immunity  to  the  action  of  the  germ. 

.NH.CH3 
Methyl  guanidin,  CaH^Na ,    NH  =  C  /  .    This  base  has  long 

been  known  as  a  product  of  the  oxidation  of  creatin  and  crea- 
tinin,  but  has  never  been  met  with  in  animal  tissues.  Brieger 
in  1886  (III.,  33)  obtained  it  from  horseflesh  which  was  al- 
lowed to  decompose  in  a  closed  vessel  at  a  low  temperature  (—9° 
to  -1-5°)  for  four  months.     Bocklisch  {Ber.,  20,  1441)  isolated  it 


METHYL  GUANIDIN.  283 

from  impure  cultures  on  beef-broth  of  Finkler  and  Prior's  vibrio 
proteus,  containing  ordinary  putrefaction  bacteria,  for  twenty  to 
thirty  days  at  37°- 38°.  Vibrio  proteus  alone  seems  incapable  of 
forming  this  base.  The  comma  bacillus,  after  some  time  (six  weeks), 
partially  decomposes  creatinin  with  formation  of  a  small  quantity  of 
methyl  guanidin  (Brieger).  The  bacillus  of  anthrax  likewise  is  cap- 
able of  transforming  creatin  into  methyl  guanidin.  It  has  been 
found  in  rabbits  that  died  of  rabbit  septicemia. 

It  occurs  in  the  mercuric  chlorid  filtrate  (Brieger),  from  which  it 
is  obtained,  after  the  removal  of  the  mercury  by  hydrogen  sulphid, 
on  precipitation  with  phosphomolybdic  acid.  The  precipitate  is  de- 
composed with  neutral  lead  acetate,  and  the  filtrate  from  this,  after 
removal  of  the  lead  by  hydrogen  sulphid,  is  concentrated  and  then 
sodium  picrate  added.  The  resinous  picrate  precipitate  is  purified  by 
boiling  with  much  water,  and,  finally,  it  is  recrystallized  from  boil- 
ing absolute  alcohol.  According  to  Bocklisch,  it  occurs  in  the  mer- 
curic chlorid  precipitate  (not  in  the  filtrate),  from  which  it  is  isolated, 
after  removal  of  the  mercury  and  concentration  of  the  clear  filtrate, 
by  precipitation  with  sodium  picrate.  The  precipitate,  containing 
cadaverin,  methyl  guanidin,  and  creatinin,  is  boiled  with  absolute 
alcohol  (cadaverin  picrate  is  insoluble)  and  the  alcoholic  solution  is 
then  evaporated  to  drive  off  the  alcohol  and  taken  up  with  water. 
From  this  aqueous  solution,  after  removal  of  picric  acid,  methyl 
guanidin  is  precipitated  by  gold  chlorid  whereas  creatinin  remains 
in  solution. 

This  ptomain  is  identical  with  the  synthetic  methyl  guanidin 
(methyluramin),  which  can  be  readily  obtained  by  boiling  a  creatin 
solution  with  mercuric  oxid  or  with  lead  dioxid  and  dilute  sul- 
phuric acid  (Dessaignes).  The  parent  substance  of  methyl  guan- 
idin as  it  occurs  in  putrefaction  is  undoubtedly  the  creatin  which 
exists  preformed  in  the  muscular  tissue.  If  such  is  the  case,  the 
bacteria  engaged  in  its  production  must  be  considered  as  possessing 
an  oxidizing  action,  since  this  base  is  prepared  synthetically  from 
creatin  by  oxidation.  The  change  would  correspond  to  that  observed 
in  the  putrefaction  of  ornithin  and  lysin  whereby  putrescin  and  ca- 
daverin result  (p.  268).  The  methyl  guanidin  in  turn  may  be  con- 
verted into  methyl  urea  and  this  into  ammonia  and  methylamin. 
That  creatin  does  not  offer  much  resistance  to  the  action  of  bacteria 
is  shown  in  the  fact  that  Friedlander's  pneumococcus,  which 
possesses  but  small  chemical  powers,  is  capable  of  slowly  but  stead- 
ily decomposing  creatin,  yielding  as  one  of  the  products  acetic  acid. 
Strecker  and  Erlenmeyer,  as  well  as  Baumann,  have  shown  that 
creatin,  although  a  substituted  guanidin,  is  not  poisonous,  but  is 
readily  converted  into  creatinin  which  is  a  relatively  toxic  sub- 
stance. On  the  other  hand,  guanidin  and  methyl  guanidin  are  quite 
violent  poisons.     This  is,  therefore,  another  instance  in  which  a  toxic 


284  CHEMISTRY  OF  THE  PTOMAINS. 

substance  is  formed  by  the  action  of  bacteria  upon  a  previously  non- 
poisonous  base  (see  page  290).  According  to  Lossen,  guanidin  is 
formed,  though  in  small  quantity,  in  the  oxidation  of  albumin. 

Fischer  (1897)  obtained  this  base  by  oxidizing  1-7  di-methyl 
guanin  whereas  7  methyl  guanin  gave  only  guanidin.  It  would 
appear  from  this  that  the  purin  antecedent  may  constitute  a  source 
of  methyl  guanidin  and  even  of  creatinin.  The  hexon  base  arginin 
(which  see)  contains  a  guanidin  group  and  either  it  or  a  related 
body  may  likewise  share  in  the  production  of  these  bodies. 

The  formulas  of  these  closely  related  substances  are  here  given  for 
comparison : 


Guanidin.  Urea. 

NH  =  C<  0  =  C< 


■-< 


Methyl  Guanidin.  Methyl  Urea. 

/NH.CH3  /NH-CH, 

NH  =  C<  0  =  C< 

Creatinin.  Methyl  Hydantoin. 

/N(CH3).CH2  /N(CH3).CHj 

NH  =  C<  I  0  =  C<  I 

\NH   —  CO  ^NH  —  CO 

Creatin.  Methyl  Hydantoic  Acid. 

/N(CH3^.CH2.C02H  /N(CH,).CH2.C0,H 

NH  =  C<  0  =  C< 

Methyl  guanidin  forms  a  colorless,  easily  deliquescent  mass  pos- 
sessing a  strong  alkaline  reaction.  On  heating  with  potassium 
hydrate  it  decomposes  and  yields  ammonia  and  methylamin.  It  is 
a  highly  poisonous  base. 

The  hydrochlorid,  C2HyN3.IICl,  can  be  obtained  from  the  picrate 
by  dissolving  the  latter  in  water  acidulated  with  hydrochloric  acid, 
and  extracting  the  solution  with  ether  to  remove  the  picric  acid. 
The  colorless  aqueous  solution  now  on  evaporation  yields  a  thin 
syrup  which  crystallizes  in  vacuum  to  compact  prisms.  These  are 
insoluble  in  alcohol,  and  give  with  platinum  chlorid  a  double  salt  of 
monoclinic  needles  (Haushofer)  which  are  very  easily  soluble  (1  part 
in  about  7  parts  of  water,  Tatarinow). 

The  aurochlorid,  C^H^Ng.HCl.AuCls  (Au  =  47.71  per  cent.),  forms 
rhombic  crystals  (Haushofer)  which  are  easily  soluble  in  ether,  more 
difficultly  in  water  or  alcohol ;  readily  soluble  (Brieger).  It  readily 
decomposes  on  heating  in  pure  water,  but  may  be  crystallized  from 
water  acidulated  with  hydrochloric  acid.     It  melts  at  198°. 

The  picrate,  C2H^N3.CgH2(N02)30II,  comes  down  at  first  as  a 
resinous  precipitate,  which  when  boiled  with  much  water  solidifies  in 
the  form  of  felted  needles.     It  is  very  difficultly  soluble  in  water, 


MOBBHUIN.  285 

and  can  be  purified  by  repeated  recrystallization  from  boiling  abso- 
lute alcohol — distinction  from  cadaverin.  It  melts  at  192°.  Ac- 
cording to  Fischer  it  forms  long  yellow  plates  which  melt  at  200° 
and  decompose  with  evolution  of  gas  at  250°. 

The  oxalate,  (C2H7N3)2.H2C20^  +  2H2O,  forms  crystals  which  are 
easily  soluble  in  water. 

Methyl  guanidin  as  obtained  from  putrefying  flesh  is  identical  in 
its  physiological  action  with  the  synthetic  base.  It  has  already  been 
stated  that  the  non-poisonous  creatin  is  readily  converted  into  the 
relatively  energetic  poison  creatinin.  The  latter  substance  possesses 
a  paralyzing  action  differing  very  much  from  its  decomposition  prod- 
uct methyl  guanidin.  This  base  is  very  poisonous,  and  the  symp- 
toms are  marked  by  dyspnoea,  muscle-tremor,  and  general  clonic 
convulsions.  Brieger  has  observed  the  following  symptoms  on  in- 
jection of  about  0.2  gram  of  methyl  guanidin  into  a  guinea-pig : 
The  respiration  at  once  becomes  more  rapid,  and  in  a  few  minutes 
abundant  passage  of  urine  and  stool  takes  place ;  the  pupils  dilate 
rapidly  to  the  maximum  and  cease  to  react.  The  animal  is  uneasy 
but  motionless,  though  not  exactly  paralyzed.  Respiration  becomes 
deeper  and  more  labored,  the  head  moves  from  side  to  side,  the  ex- 
tremities become  gradually  paralyzed ;  dyspnoea  sets  in,  the  animal 
falls  on  its  side,  and  dies  (twenty  minutes)  amid  general  clonic  con- 
vulsions of  short  duration.  Fibrillary  twitchings  of  the  trunk- 
muscles  are  observed  only  in  the  beginning.  Post-mortem  showed 
the  heart  to  be  stopped  in  diastole,  the  intestines  filled  with  fluid, 
the  bladder  contracted,  the  cortex  of  the  kidney  hypersemic,  but  the 
papillae  of  the  kidneys  surprisingly  pale. 

Morrhuin,  Ci9H27N3,  was  obtained  by  Gautier  and  Mourgues 
(1888)  from  the  mother-liquors  of  asellin  on  concentration  of  the 
platinum-containing  liquid.  This  substance  constitutes  about  one- 
third  (0.07  per  cent.)  of  all  the  bases  found  in  cod-liver  oil,  and  is 
named  after  Gadus  morrhua,  the  ordinary  codfish.  The  free  base  is 
an  oily,  very  thick,  amber-yellow  liquid,  the  odor  of  which  resem- 
bles somewhat  that  of  syringa.  It  floats  on  water  and  partially  dis- 
solves ;  is  more  soluble  in  ether  and  in  alcohol.  The  base  is  very 
alkaline  and  is  caustic  to  the  tongue.  It  absorbs  carbonic  acid  and 
is  non-volatile.  The  salts  of  copper  are  precipitated  by  it  but  the 
hydrate  formed  is  not  redissolved. 

The  hydrochlorid  is  very  deliquescent.  The  gold  salt  forms  a 
yellow  precipitate  which  readily  dissolves  on  warming.  The  plati- 
num salt,  Ci9ll27N3.2HCl.PtCl^  (Pt  =  27.56  per  cent.),  crystallizes 
in  barbed  needles,  which  are  quite  soluble.  (Separation  from  asel- 
lin, p.  286.) 

The  base  possesses  the  property  of  exciting  the  appetite ;  it  acts 
as  a  diaphoretic,  and,  above  all,  as  a  diuretic.     0.029  gram  given 


286  CHEMISTRY  OF  THE  PTOMAINS. 

subcutaneously  to  a  guinea-pig  produced  in  two  and  a  half  hours  a 
loss  of  13.5  grams  in  the  weight  of  the  animal.  The  same  effect  is 
produced  in  birds.  Strong  doses  (0.1  gram  per  kilogram)  produce 
fatigue  and  hebetude. 

A  Base,  C^gHg^N^,  was  obtained  as  early  as  1868  by  Oser,  who 
observed  its  formation  during  the  fermentation  of  pure  cane-sugar 
by  means  of  yeast.  The  hydrochlorid  when  dried  in  vacuo  is  said 
to  form  a  white,  very  hygroscopic  foliaceous  mass  which  soon  be- 
comes brown  on  exposure  to  air.  At  first  it  imparts  a  burning  taste 
which  is  soon  replaced  by  a  very  bitter  sensation. 

A  Base  corresponding  to  the  formula  Cj^yH^gN^  was  obtained  by 
Gautier  and  Etard  from  the  mother-liquors  of  the  platinochlorid  of 
the  base  CgHjgN.  Very  little  is  known,  however,  in  regard  to  the 
general  properties  of  this  base  owing  to  the  small  quantity  which 
could  be  isolated.  This  base  and  the  one  obtained  by  Oser  from  the 
yeast  fermentation  of  sugar,  Cj3H2oN4 ,  and  asellin,  CjgHggN^,  are  the 
only  ptomains  thus  far  isolated  which  are  known  to  contain  four 
atoms  of  nitrogen. 

The  platinochlorid,  Ci^H3gN,.2HCl.PtCl4(Pt  =  27.52  percent.), 
is  readily  soluble  and  crystallizes  in  needles  which  possess  a  light- 
yellow  flesh  color.  When  heated  to  100°,  it  slowly  decomposes 
giving  off  a  syringa-like  odor. 

Asellin,  Q^^^^^,  was  isolated  by  Gautier  and  Mourgues  (1888), 
together  with  five  other  bases,  from  cod-liver  oil.  It  is  present  only 
in  small  quantity  in  the  oil.  The  name  is  derived  from  Asellus 
major,  the  great  codfish.  The  free  base  is  thrown  down  from  the 
solutions  of  the  hydrochlorid  by  the  addition  of  alkali,  in  amorphous 
white  floccules  which  are  almost  insoluble  in  water.  It  is  almost 
colorless,  but  on  exposure  to  the  air  becomes  slightly  green.  It  is 
not.  hygroscopic,  and  possesses  a  density  of  about  1.05.  On  heating 
it  melts  to  a  viscid  yellowish  fluid,  possessing  an  aromatic  odor ;  it 
is  non-volatile.  Although  almost  insoluble  in  water,  it  imparts  to  this 
an  alkaline  reaction  and  a  bitter  taste.  It  is  soluble  in  ether,  more 
so  in  alcohol. 

The  salts  are  crystallizable,  but  are  partially  dissociated  by  the 
action  of  warm  water.  The  hydrochlorid  forms  crossed  or  entangled 
needles  which  are  quite  bitter.  The  gold  salt  is  very  reducible. 
The  platinochlorid,  C25H32N,.2HCl.PtCl,  (Pt  =  24.41),  is  orange- 
yellow  in  color ;  soluble  in  warm  water,  insoluble  in  cold  water 
(separation  from  morrhuin,  p.  285),  and  is  rapidly  changed  by  boil- 
ing water.  The  mercury  salt  is  precipitated  in  the  cold,  redissolves 
on  lieating,  and  then,  on  cooling,  recrystallizes. 

In  large  doses  it  produces  fatigue,  short  and  rapid  respiration, 
and  stupor.  Three  milligrams  of  the  hydrochlorid  killed  a  green- 
finch in  fourteen  minutes. 


MYDIN.  287 

Mydin,  CgHj^NO,  is  a  non-poisonous  base  which  was  obtained  by 
Brieger  in  1886  (III.,  25)  from  the  putrefaction  of  about  two 
hundred  pounds  of  human  internal  organs  ;  and  also  in  cultures  of 
the  Eberth  bacillus  on  peptonized  blood-serum.  It  occurs  in  the 
mercuric  chlorid  filtrate  and  is  isolated,  after  the  removal  of  the 
mercury  by  hydrogen  sulphid,  by  precipitation  with  phosphomo- 
lybdic  acid.  The  gummy  precipitate  which  is  produced  is  de- 
composed on  the  water-bath  with  a  solution  of  neutral  lead  acetate, 
and  the  filtrate  on  evaporation  yields  a  colorless  hydrochlorid,  crys- 
tallizing in  plates.     It  is  purified  by  recrystallization  of  the  picrate. 

The  free  base  is  strongly  alkaline,  and  possesses  an  ammoniacal 
odor.  It  is  characterized  by  its  strong  reducing  properties.  The 
name  mydin  is  derived  from  ftuddw,  to  putrefy.  With  platinum 
chlorid  it  gives,  after  a  time,  an  extremely  soluble  salt ;  with  gold 
chlorid,  a  precipitate  of  metallic  gold.  On  distillation  it  is  decom- 
posed. 

The  hydrochlorid,  CgIIj,NO.HCl,  crystallizes  in  colorless  plates. 
It  gives  a  blue  color  with   ferric  chlorid  and  potassium  ferrocyanid. 

The  picrate,  CgHuNO.CgH2(N02)30H,  is  obtained  in  broad  prisms, 
which  melt  at  195°.     It  is  the  only  salt  suitable  for  manipulations. 

In  describing  Nencki's  collidin  (page  256)  it  was  stated  that 
tyrosin  might  be  looked  "  upon  as  the  source  of  that  base.  It  would 
seem,  however,  to  be  more  appropriately  the  parent  substance  of 
mydin,  inasmuch  as  it  decomposes  on  being  heated  to  270°  into  car- 
bonic acid  and  oxyphenyl-ethylamin,  CgH^^NO.  The  change  that 
takes  place  can  be  represented  by  the  equation  : 


/OH  /OH 

CeH/  =  C«h/  -f-  CO,. 

\CH,.CHNH3.C02H  ^CHj.CHj.NH^ 

Tykosin.  Oxyphenyl-ethylamin. 

A  Base,  CgHj^NO^ ,  was  isolated  by  E.  and  H.  Salkowski  (1883) 
from  decomposing  fibrin  and  meat.  An  amino-valerianic  acid  is 
formed  in  the  hydrolytic  cleavage  by  means  of  acids  of  the  protamin 
clupein  (Kossel) ;  and  of  casein  (Fischer,  1901).  The  ^-acid  also 
results  in  the  oxidation  of  piperidin  {Ber.,  25,  2778).  For  diamino- 
valerianic  acid,  see  ornithin. 

It  is  extremely  soluble  in  water,  very  difficultly  so  in  alcohol,  in- 
soluble in  ether,  and  possesses  a  semen-like  odor  and  saline  taste. 
The  aqueous  solution,  which  is  not  alkaline  in  reaction,  yields  on 
evaporation  a  stellate  crystalline  mass  which  on  standing  over  sul- 
phuric acid  becomes  a  white  powder  melting  at  156°.  It  dis- 
solves silver  oxid  but  not  cupric  hydrate,  thus  apparently  indicating 
that  it  is  not  an  amido  acid.  Moreover,  it  does  not  give  a  pre- 
cipitate or  blue  coloration  with  copper  acetate  or  ammoniacal  silver 
nitrate.  It  thus  differed  from  the  then  known  amido-valerianic  acids, 
its  isomers.     Later,  however  (1891),  Gabriel  and  Aschan  showed 


288  CHEMISFBY  OF  THE  PTOMAINS. 

that  o-amido- valerianic  acid  agrees  with  this  base  in  its  reactions  to 
copper  nitrate.  The  gold  salt  of  the  synthetic  base  possessed  the 
same  composition  as  that  of  Salkowski,  and  melted  at  86°- 87°. 

The  identity  of  this  base  with  o-amido- valerianic  acid  (homo- 
piperidinic  acid)  would  seem  to  be  established,  and  as  such  it  is 
regarded.     Its  structure,  then,  is  represented  by : 

NH2.CH2.CHi.CH2.CH2.CO2H. 

For  its  synthetic  preparation,  see  Ber.,  24,  1365  (1891).  The  base 
does  not  seem  to  possess  toxic  action. 

The  hydrochlorid,  CgHjjNOj.HCl,  forms  colorless,  stellate  crys- 
tals, which  are  permanent  in  the  air,  and  are  extremely  soluble  in 
water,  even  in  absolute  alcohol. 

The  aurochlorid,  C.B[^^N02.HCl.AuCl3  +  Hp,  is  obtained  on  slow 
evaporation,  as  large,  well  formed,  beautiful  dark  yellow  crystals. 
They  are  probably  monoclinic,  contain  water  of  crystallization  and 
melt  at  below  100°. 

The  platinochlorid  gave  on  analysis  results  corresponding  to  the 
formula  (CyHjgN02.HCl)2PtCl4.  This  possibly  may  have  been  due 
to  the  presence  of  some  higher  horaologues  of  the  base  CgH^^NOg.  It 
forms  fine  orange-yellow  crystals,  which  are  very  difficultly  soluble 
in  alcohol,  easily  so  in  hot  water,  from  which,  on  cooling,  it  crystal- 
lizes in  beautiful  plates. 

Cholin  Group. — The  following  four  bases  are  closely  related,  and, 
indeed,  starting  from  cholin,  the  oldest  and  best  known  individual, 
the  remaining  bases  can  be  readily  prepared  from  it.  Moreover, 
they  can  all  be  prepared  synthetically  according  to  methods  that 
will  be  subsequently  indicated.  As  cholin  is  the  most  prominent 
member,  we  have  thought  best  to  class  these  substances  together  as 
constituting  the  cholin  group.  It  is  very  probable  that  mydatoxin 
and  mytilotoxin,  when  their  constitution  becomes  known,  will  be 
found  to  be  homologues  of  certain  members  of  this  group. 

Neurin,  C^H^gNO  =  C2H3.N(CH3)3.0H.— This  substance  was  ob- 
tained and  named  thus  by  Liebreich  (1865),  who  prepared  it  by 
boiling  protagon  for  twenty-four  hours  with  concentrated  baryta. 
Previous  to  its  discovery  as  a  decomposition  product  of  protagon 
from  the  brain  it  was  prepared  synthetically  by  Hoffmann  (1858) 
by  treating  trimethylamin  and  ethylene  bromid  with  potassium  hy- 
drate or  silver  oxid.  Baeyer  (1866),  by  boiling  an  alcoholic  ex- 
tract of  the  brain  with  baryta  water,  obtained,  on  separation  by 
three  different  methods,  a  base  or  rather  a  mixture  of  bases,  which 
on  analysis  gave  results  corresponding  to  the  three  formulae  : 

1  2  3  •^ 

(CjH^NOCOjPtCl,        (C5Hi2NCl)2PtCl,        (C6Hi,NCl)jPtCli 


NEURIN.  289 

Formula  No.  3  was  the  one  accepted  by  Liebreich  for  neurin,  but, 
according  to  Baeyer,  Liebreich's  neurin  salt  was  not  simple  but  a 
mixture  of  Xos.  1  and  2.  He  himself  accepted  formula  No.  1  as 
the  platinochlorid  of  neurin,  and  distinctly  states  {Annal.  d.  Chera. 
u.  Pharm.,  142,  323,  1867)  that  neurin  is  in  composition  trimethyl 
oxyethyl-ammonium  hydroxid.  And  according  to  him,  cholin  from 
bile  and  sinkalin  from  white  mustard  appear  to  be  identical  with 
neurin. 

This  nomenclature  of  Baeyer's  was  at  first  adopted  by  Wurtz 
and  others,  who  showed  that  the  oxy ethyl  base  was  identical  with 
cholin  and  sinkalin.  On  that  account  Strecker,  in  1868  [Annal., 
148,  79),  suggested  the  restriction  of  the  name  cholin  to  the  oxy- 
ethyl  base,  and  to  reserve  the  name  neurin  for  the  base  whose  platin- 
ochlorid is  represented  in  No.  3,  as  originally  was  done  by  Lieb- 
reich. In  1869  Liebreich  showed  that  pure  protagon,  when  heated 
with  baryta  for  twenty-four  hours,  yields  a  substance  having  the 
composition  of  the  vinyl  base  : 

r  (CH3), 

N  -^  CH  =  CHj 
(OH. 

The  platinochlorid  of  this  base  crystallized  in  five-sided  yellow 
plates,  which  after  a  time,  on  exposure  to  the  air,  became  cloudy  ; 
on  treatment  now  with  water  a  portion  dissolved  and  the  solution 
was  found  to  contain  the  oxyethyl  base.  Furthermore,  he  observed 
that  when  the  alcoholic  extract  of  the  brain,  from  which  all  the  pro- 
tagon had  been  removed,  is  treated  with  baryta,  only  the  latter,  the 
oxyethyl  base,  is  obtained.  Finally,  in  1870,  Wurtz  abandoned  the 
use  of  the  term  neurin  to  designate  the  oxyethyl  base,  and  returned 
to  the  name  cholin,  originally  applied  to  the  oxyethyl  base  by  its 
discoverer,  Strecker.  Nevertheless,  the  confusion  in  the  use  of  these 
two  terms  continued  to  exist  even  until  very  recent  years,  causing  no 
little  misunderstanding.  Thus,  Marino-Zuco  (1885),  in  his  excel- 
lent researches  on  the  genesis  of  ptomai'ns,  applies  the  term  neurin, 
following  Baeyer's  precedent,  to  the  oxyethyl  base,  C^Hj^NOg ,  which 
is  really  cholin,  according  to  the  proper  nomenclature. 

We  have  gone  somewhat  at  this  point  in  detail  into  the  history 
and  proper  use  of  the  terms  neurin  and  cholin  because  of  the  confu- 
sion which  is  sure  to  arise  if  the  distinction  is  not  thoroughly  borne 
in  mind.  The  name  neurin,  then,  should  be  used  only  to  denote  the 
vinyl  base  C^HjgNO.  It  is  trimethyl-vinyl-ammonium  hydrate. 
On  the  other  hand,  cholin  is  the  oxyethyl  base  CgHj^NO^ ,  which  is 
trimethyl-oxyethyl-ammonium  hydrate. 

Neurin  has  been  obtained  by  Brieger  (1883)  in  the  putrefaction  of 

horse,  beef  and  human  flesh  for  five  or  six  days  in  summer.     It  also 

occurs  in  the  commercial,  so-called  "neurin,"  together  with  cholin 

(Brieger,   I.,  34) ;    in   commercial    25   per  cent,  cholin  (Schmidt). 

19 


290  CHEMISTRY  OF  THE  PTOMATNS. 

Liebreich  obtained  it  in  the  decomposition  of  protagon  by  baryta. 
Gulewitsch  (1899),  however,  was  unable  to  confirm  this  change,  nor 
was  he  able  to  detect  neurin  in  fresh  brain  matter.  Liebreich's  re- 
sult may  possibly  be  due  to  the  use  of  partly  decomposed  organs,  in 
which  neurin  would  be  present  as  a  result  of  the  bacterial  cleavage  of 
cholin.  Brieger  (I.,  60)  also  has  isolated  it  along  with  cholin 
from  fresh  human  brains  by  boiling  with  baryta,  but  did  not  obtain 
it  by  digesting  the  brains  on  the  water-bath  with  2  per  cent,  hydro- 
chloric acid.  It  has  been  found  in  putrid,  and  as  a  result  of  this 
change,  poisonous  mushrooms  (Berlinerblau,  1888).  For  its  syn- 
thetic preparation,  see  Gulewitsch  (Zeitsoh.  /.  physiol.  Chem.,  26,  175). 

The  genesis  of  neurin  is  still  rather  obscure,  and  it  is  to  be  hoped 
that  future  investigations  may  shed  more  light  upon  the  mysterious 
production  of  this  highly  poisonous  base.  Its  occurrence  in  the 
brain  together  with  cholin  would  seem  to  indicate  that  it  is  either 
derived  from  cholin  by  the  removal  of  water,  or  that  it  exists  to- 
gether with  cholin,  partly  replacing  the  latter  in  the  molecule  of 
protagon  (lecithin),  according  to  the  hypothesis  put  forward  by 
Lippmann  (page  297). 

The  question  of  its  derivation  from  cholin  by  withdrawal  of  a 
molecule  of  water  was  subjected  early  to  an  interesting  experimental 
discussion.  Ch.  Gram  attempted  to  explain  the  production  of  neu- 
rin and  other  muscarin-like  ptomains  as  due  to  the  dehydrating  ac- 
tion of  the  acids  employed  in  the  methods  of  extraction,  and,  indeed, 
he  claimed  to  have  converted  cholin  platinochlorid,  by  heating  with 
hydrochloric  acid,  into  neurin.  This  statement  was  disputed  by 
Brieger,  and  by  others,  who  showed  that  the  platinochlorid  of  cholin, 
as  well  as  the  hydrochlorid,  may  be  heated  with  fifteen  or  thirty  per 
cent.,  or  even  concentrated  hydrochloric  acid,  for  six  to  eight  hours 
on  a  water-bath  without  any  conversion  whatever  (III.,  15). 

That  neurin  may  be  obtained  from  cholin,  at  least  by  chemical 
processes,  was  shown  by  Baeyer,  in  1866,  who  found  that  cholin 
chlorid,  when  heated  with  several  times  its  volume  of  concentrated 
hydriodic  acid  and  some  red  phosphorus,  gave  a  compound  CglljgNIj 
which,  on  digestion  with  fresh,  moist  silver  oxid,  yielded  a  vinyl 
base  identical  with  that  previously  obtained  synthetically  by  Hof- 
mann  and  now  known  as  neurin.  In  Hofmann's  method  for  the 
synthesis  of  neurin  the  trimethylamin  ethylene  bromid  (see  synthesis 
of  cholin,  p.  296)  is  treated  with  fresh  moist  silver  oxid.  Schmidt 
and  Bode  have  shown  that  the  iodin  compound  resulting  from  the 
action  of  hydriodic  acid  on  cholin  is  the  same  as  that  formed  by  the 
action  of  the  acid  on  neurin  ;  and  that  on  treatment  with  silver  oxid 
it  yields  neurin.  Cholin,  therefore,  may  be  readily  changed  into 
neurin.  On  the  other  hand,  since  neurin  with  hydriodic  acid  forms 
the  same  compound,  by  heating  with  silver  nitrate  cholin  is  tformed. 
Hence  neurin  can  be  easily  re-converted  into  cholin  (Schmidt). 


NEUBIN.  291 

Ou  distilling  cholin  with  water,  or  on  dry  distillation,  a  little 
neurin  forms  (Nothnagel).  Brieger  has  tried,  unsuccessfully,  to 
bring  about  this  dehydration  by  the  putrefaction  of  pure  cholin  (I., 
59).  However,  Schmidt  and  Weiss  (1887)  were  more  successful, 
and  they  found  that  cholin,  as  well  as  the  hydrochlorid  and  lactate, 
is  changed  by  the  action  of  microorganisms  into  the  strongly  poison- 
ous neurin.  Their  results  are  given  in  full  under  cholin  (see  page 
299).  More  recently  (1890)  Nesbitt  in  his  study  upon  auto-intoxi- 
cations endeavored  to  show  that  the  toxic  neurin  may  form  in  the 
intestines  by  the  dehydration  of  the  cholin  in  lecithin.  After  feed- 
ing dogs  on  yolk  of  eggs  for  several  days,  the  intestines  were  ligated 
and  eventually  the  contents  were  examined.  Cholin  was  found,  like- 
wise good  evidence  of  neurin,  and  a  ptomain  mentioned  on  p.  260. 
From  what  has  been  said  it  is  evident  that  neurin  can  only  arise 
from  cholin,  and  this,  as  will  be  seen  later,  is  derived  from  lecithin. 
Cholin  may  be  kept  dry  or  in  aqueous  solution  for  four  months 
without  change  into  neurin  (Schmidt). 

Neurin  is  almost  invariably  accompanied  by  cholin,  from  which, 
however,  it  can  be  readily  separated  by  the  difference  in  the  solubil- 
ities of  the  platinochlorids.  It  occurs  in  the  mercuric  chlorid  pre- 
cipitate (and  in  the  filtrate),  and  from  this  it  can  be  obtained,  after 
removal  of  the  mercury,  by  precipitating  the  solution  of  the  mixed 
hydrochlorids  in  absolute  alcohol  by  platinum  chlorid.  The  platino- 
chlorids are  then  separated  by  recrystallization  from  water,  since  the 
neurin  is  difficultly  soluble,  while  the  cholin  salt  is  readily  soluble. 

The  free  base  possesses  a  strong  alkaline  reaction,  and  on  contact 
with  the  fumes  of  hydrochloric  acid  it  yields  a  cloud.  It  liberates 
ammonia  from  its  salts  even  in  the  cold.  It  behaves  toward  the 
heavy  metals  like  a  strong  base,  and  prevents  the  coagulation  of  albu- 
min by  heat.  On  boiling  a  concentrated  solution  of  neurin  it  yields 
trimethylamin.  It  is  not  affected  by  prolonged  boiling  with  baryta 
or  with  sodium  alcoholate.  According  to  Liebreich,  the  alkaline 
solution  cannot  be  neutralized  by  passing  through  it  carbonic  acid. 

With  hydriodic  acid  neurin  at  140°- 150°  forms  trimethylamin 
ethylene  iodid,  which,  on  treatment  with  silver  nitrate,  as  stated 
above,  forms  cholin,  or  on  treatment  with  silver  oxid  regenerates 
neurin  (Schmidt).  This  compound  is  the  same  as  that  prepared  by 
Baeyer.     It  melts  at  231°. 

Fuming  hydrobromic  acid  has  no  action  on  neurin  at  ordinary 
temperature  or  at  100°,  but  at  160°- 165°  it  yields  trimethylamin- 
ethylene  bromid,  which  behaves  with  silver  nitrate  or  oxid  as  above 
(Bode  and  Schmidt).     Thus : 

(1)  Br.N(CH3)3.C,H,Br  +  2AgN03  +  H^O  =  N03.N(CH3)3.CjH,OH  +  2AgBr  + 

HNO3. 

(2)  BrN(CH3)3.C,H,Br  +  Ag,0  =  0H.N(CH,)j.CH.CH2  +  2AgBr. 


292  CHEMISTRY  OF  THE  PTOMAINS. 

This  bromin  compound  is  formed  in  the  synthesis  of  neurin  out 
of  trimethylamin  and  ethylene  bromid.  It  melts  at  230°,  is  soluble 
in  water  and  in  hot  alcohol  and  forms  stellate  masses  of  small  plates 
or  prisms.  With  freshly  precipitated  silver  oxid,  according  to 
equation  2,  it  yields  neurin. 

Hypochlorous  acid,  likewise,  breaks  up  the  vinyl  group  in  neurin 
to  form  a  derivative  of  cholin,  which  with  silver  oxid  yields  iso- 
muscarin  (Bode). 

It  decomposes  readily  on  standing,  more  rapidly  on  heating  into 
trimethylamin. 

The  chlorid,  CgHjgN.Cl,  is  extremely  poisonous  and  crystallizes  in 
fine  hygroscopic  needles.  It  is  easily  soluble  in  water  and  alcohol. 
By  the  action  of  hypochlorous  acid  it  is  changed  to  iso-muscarin. 
For  behavior  to  alkaloidal  reagents,  see  Table  I.;  also  Gulewitsch. 

The  bromid,  Br.N(CH3)3.C2H3,  is  colorless,  wart-like  in  form, 
hygroscopic  and  easily  soluble  in  water  and  in  alcohol,  insoluble  in 
ether.     It  melts  at  193°  (Bode). 

The  iodid,  IN(CH3)3.C2H3 ,  forms  colorless,  permanent  needles ; 
easily  soluble  in  water ;  slightly  in  cold  alcohol,  easily  in  hot  alco- 
hol. When  heated  to  180°  it  becomes  yellow,  and  at  196°  it  melts 
(Bode). 

The  picrate  forms  feathery,  gold-yellow,  long  needles  which  when 
heated  rapidly  melt  with  decomposition  at  263°- 264°.  At  23° 
the  solubility  in  water  is  1:91.6.  On  heating  it  dissolves  more 
readily  in  water  and  in  alcohol  (Gulewitsch). 

The  platinochlorid,  {C^B.^^.Q\)^iQ\  (Pt=  33.60  per  cent.),  is 
difficultly  soluble  in  hot  water,  and  crystallizes  in  beautiful,  well 
formed,  small  octahedra  belonging  to  the  regular  system.  The 
crystals  are  always  single.  No  twin  crystals  are  observed.  Some- 
times the  crystals  contain  water  of  crystallization,  at  other  times 
they  do  not  (Brieger,  I.,  33).  It  melts  at  211°-213°  (Schmidt); 
213°-214°  (Bode) ;  with  decomposition  at  195.5-198°  (Gulewitsch). 
According  to  Liebreich,  it  forms  from  an  aqueous  solution  in  five- 
or  six-sided,  heaped-up  plates  resembling  urea  nitrate,  while  from 
an  alcoholic  solution  it  forms  needles  which  on  exposure  to  air  be- 
come opaque  and  are  partially  converted  into  the  oxyethyl  base — 
cholin.  The  six-sided  plates  are  rarely  met  with  and  readily  show 
an  octahedral  relation  (Gulewitsch),  The  difficult  solubility  (at 
20.5°  it  is  1  :37.6  part  of  water),  octahedral  form,  always  single, 
and  the  melting-point  distinguish  it  perfectly  from  the  cholin  salt. 

The  aurochJorid,  CgHj^N.Cl.AuClg  (Au  =  46.37  per  cent.),  forms 
a  yellow  cheesy  precipitate  which  on  recrystallization  yields  flat 
prisms  which  are  difficultly  soluble  in  hot  water  (Brieger).  At 
21.5°  it  dissolves  in  336.5  parts  of  water  (Gulewitsch).  Dissolves 
easily,  and  can  be  purified  by  crystallization  (Liebreich).  It  melts 
at  228°- 232°  (Gulewitsch);   238°- 239°  (Klein). 


CHOLIN.  293 

The  mercurochlorid  may  exist  in  two  forms :  CgHjjNCl  +  GHgClg 
and  C,Hj.,NCl  +  HgCl2.  The  former  yields  colorless,  dull,  very  frag- 
ile plate-like  crystals  which  are  difficultly  soluble  and  melt  at  230°- 
234°.  The  latter  forms  aggregates  of  very  narrow,  rather  long  frag- 
ile prisms,  which  are  more  soluble  in  water  than  the  preceding  and 
melt  at  198.5°  -  199.5°  (Gulewitsch). 

Physiological  Action. — Neurin  is  exceedingly  poisonous,  even  in 
small  doses,  and  in  its  action  it  strongly  partakes  of  the  character- 
istic stamp  of  poisoning  by  muscarin.  The  injection  of  a  few  milli- 
grams into  frogs  produces  in  a  short  time  a  complete  paralysis  of 
the  extremities,  with  deadening  of  reflex  excitability.  Respiration 
stops  first,  while  the  rate  of  heart-beat  gradually  decreases  till, 
finally,  stoppage  in  diastole  takes  place.  The  injection  of  atropin 
at  this  point  does  away  with  the  effect  of  neurin,  so  that  the  heart 
begins  to  beat  again.  Previously  atropinized  frogs,  as  a  rule,  with- 
stand the  action  of  the  poison.  Immediately  after  the  introduction 
of  this  substance  there  can  be  observed  a  distinct  period  of  exalta- 
tion, which,  however,  soon  gives  way  to  the  characteristic  stage  of 
depression  seen  in  the  progressive  slowing  of  the  rate  of  heart-beat. 
Of  the  warm-blooded  animals,  cats  seem  to  be  much  more  sensitive 
to  its  action  than  mice,  rabbits  or  guinea-pigs.  The  symptoms  seen 
in  rabbits  are  profuse  moistening  of  the  nasal  cavities  and  upper 
lip,  which  is  succeeded  by  an  intensely  profuse  salivation ;  later 
on  there  is  noticeable  an  abundant  secretion  from  the  nasal  mucous 
membrane  and  from  the  eyes ;  the  latter,  however,  ceases  in  a  short 
time.  The  movements  of  the  heart  and  of  respiration  are  at  first 
quickened  and  strengthened,  but  before  long  the  paralytic  effects 
produce  a  constant  slowing  and  weakening  till  finally  complete  ces- 
sation of  both  movements  results.  The  decided  dyspnoea  observed 
gradually  alters  its  character,  and  just  before  death  the  respiration 
is  irregular  and  superficial.  The  heart,  as  in  frogs,  continues  to 
beat  after  the  respiratory  movements  have  ceased  and  finally  it 
stops  in  diastole.  Direct  application  of  concentrated  solutions  of 
the  poison  to  the  eyes  produces  almost  always  a  contraction  of  the 
pupil,  while  a  similar  but  less  constant  contraction  is  seen  when 
it  is  injected.  The  peristaltic  action  of  the  intestines  is  height- 
ened to  such  an  extent  that  continual  evacuation  takes  place.  Just 
before  death,  violent  clonic  convulsions  occur.  Atropin  pos- 
sesses a  strong  antagonistic  action  toward  neurin  and  the  injection 
of  even  a  small  quantity  is  sufficient  to  dispel  the  symptoms  just 
described. 

Cholin,  C-H^^NO,  =  C2HpH.N(CH3)3.0H.— This  base  is  iden- 
tical with  the  sinkalin  of  von  Babo,  the  bilineurin  of  Liebreich,  and 
the  neurin  of  Baeyer,  Marino-Zuco,  and  others.  According  to 
Schmiedeberg  and  Harnack,  it  is  identical  with  Letellier's  amanitin 


294  CHEMISTRY  OF   THE  PTOMAINS. 

(agaricin),  to  which  they  assign,  however,  the  formula  (CH3)3N. 
(CHOH.CH3)OH. 

Cholin  was  first  prepared  and  so  named  by  Strecker,  1862,  by 
treating  hog-bile  with  hydrochloric  acid.  It  was  prepared  synthet- 
ically by  Wurtz  (1868)  by  direct  union  of  ethylene  chlorhydrin  and 
trimethylamin.  The  reaction  that  takes  place  can  be  represented 
by  the  equation  : 

c,H,{<^H  ^     ju^     gin  NCI. 

^^^^^  CjH^.OH  J 

Baeyer  (1866)  obtained  it  by  boiling  an  alcoholic  extract  of  the 
brain  with  baryta  water;  and  Liebreich,  in  1869,  showed  that  if 
the  alcoholic  extract  from  which  all  the  protagon  had  been  re- 
moved be  thus  treated  only  cholin  is  formed,  whereas  pure  prota- 
gon on  heating  with  baryta  yields  neurin  (not  confirmed  by  Gule- 
witsch).  It  has  been  obtained  from  the  yolk  of  eggs ;  from  bile ; 
from  fresh  brains  (Brieger)  ;  from  fresh  eggs,  blood,  lungs,  and 
hearts,  and  from  lecithin  (Marino-Zuco)  ;  from  human  placenta 
(Bohm) ;  from  the  eye ;  from  commercial  neurin  (Brieger) ;  neurin 
was  found  in  a  specimen  of  commercial  cholin  (Schmidt)  ;  in  com- 
mercial muscarin  sulphate  (Nothnagel)  ;  from  fresh  as  well  as  de- 
composing internal  organs  of  the  cadaver  (Brieger,  1885) ;  in  fresh 
blood  (neurin  of  Marino-Zuco  and  Martini) ;  from  herring-brine 
and  decomposing  pike,  three  days  in  midsummer  (Bocklisch).  It 
has  also  been  isolated  from  cultures  of  vibrio  pro  tens  (Bocklisch, 
Carbone)  and  of  comma  bacillus  (Brieger).  Ehrenberg  (1887) 
found  it  in  poisonous  sausages,  and  by  growing  a  bacillus  obtained 
from  this  on  liver.  Gulewitsch  has  isolated  cholin  from  horseflesh, 
putrefying  at  15°  for  four  months,  together  with  cadaverin  and 
probably  muscarin.  It  has  been  found  in  the  intestines  of  dogs 
together  with  neurin  and  another  ptomain  (p.  260)  (Nesbitt)  ;  and 
in  fermented  fish  together  with  various  amines  (Morner). 

According  to  Halliburton  (1901)  cholin  can  be  extracted  from 
fresh  brains  by  means  of  physiological  salt  solution.  The  fact  that 
this  base  readily  forms  in  the  disintegration  of  nerve  tissue  has  led 
to  the  belief  that  it  may  be  a  most  important  factor  in  auto-intoxi- 
cations. Halliburton  has  found  cholin  in  traces  in  the  cerebro-spinal 
fluid  and  in  the  blood  in  nerve  degenerations  and  during  the  convul- 
sive seizures  in  general  paralysis  of  the  insane,  and  hence  he  regards 
this  condition  as  probably  due  to  cholin  poisoning  although  other 
products  may  be  also  present.  In  regard  to  the  possible  conversion 
of  cholin  into  neurin  and  the  presence  of  the  latter  in  auto-intoxi- 
cations, see  pages  291  and  299.     See  also  Florence's  crystals. 

Not  only  has  cholin  been  met  with  in  the  animal  tissues',  but  it 
has  also  been  observed  within  the  last  few  years  to  be  very  widely 


CHOLIN.  296 

distributed  in  the  vegetable  kingdom,  especially  so  in  fatty  seeds. 
Thus  it  has  been  found  (Harnack,  1876)  accompanying  muscarin  in 
toadstool  (Agaricus  muscarius) ;  in  bops,  and  hence  in  beer  (Griess 
and  Harrow) ;  in  the  seeds  of  Trigonella,  in  Indian  hemp,  areca- 
and  earth-nuts,  hemp  seeds  and  lentils  (Jahns) ;  in  the  seeds  of  white 
mustard,  as  a  glycosid  (von  Babo) ;  in  ergot  (Brieger)  ;  in  the  germs 
of  pumpkins  and  lupines  (Schulze,  Zeitsehr.  f.  physiol.  Ghem.,  11, 
365) ;  in  beech-nuts  and  morels  (Helvella  esculenta.  Boletus  luridus, 
Amanita  pantherina,  Bohm)  ;  in  flores  sambuci  (elder),  and  extracts 
of  belladonna,  hyoscyamus,  ipecacuanha  root  and  Acorus  calamus 
(Kunz),  ipecacuanha  root  (Arndt),  and  Scopolia  Japonica  (Schmidt 
and  Henschke) ;  in  the  sprouts  and  cotyledons  of  Sqja  beans  (Schulze, 
1888),  in  the  fat  from  hog's  bean,  vetch,  peas  and  lupines  (Jacobson, 
1889) ;  from  the  lecithin  of  lupine  seeds  (Schulze  and  Steiger)  ;  and 
in  Cheken  leaves  (Myrtus  cheken,  AVeiss).  According  to  Lippmann 
(Ber.,  20,  3206),  it  is  present  together  with  betain  in  the  molasses 
from  beet-root  sugar.  Cholin  (Ritthausen)  and  betain  (Bohm)  exist 
together  in  cotton-seeds ;  hence,  cholin  occurs  in  the  press-cakes 
from  cotton-seeds  (Bohm).  Maxwell  by  extraction  of  cotton-seed 
cake  with  alcohol  obtained  about  five  times  as  much  betain  as  cholin. 
With  betain  it  occurs  in  worm-seed  (Artemisia  Cina,  Jahns) ;  in 
sprouts  of  malt  and  wheat  (Schulze  and  Frankfurt).  According  to 
Schulze,  and  also  Ritthausen,  cholin  occurs  with  betain  and  another 
base  in  the  seed  of  the  vetch,  and  in  peas  with  a  base  resembling 
betain.  The  two  bases  have  also  been  found  together  in  the  roots 
and  leaves  of  Scopolia  atropoides  by  Siebert. 

Partheil  found  cholin,  but  not  betain,  in  the  seeds  of  Cytisus 
laburnum.  Kresling  obtained  it  from  the  pollen  of  the  fir,  Pinus 
sylvestris. 

Schulze  and  his  pupils  have  shown  that  argiuin,  cholin,  and  xan- 
thin  bases  occur  in  lupine  sprouts.  The  same  compounds  with  ver- 
nin  occur  in  gourd  sprouts,  whereas  in  the  sprouts  of  Vicia  sativa, 
betain,  cholin,  and  guanidin  are  present.  The  latter  is  undoubtedly 
derived  from  arginin,  which  see. 

Cholin  may  readily  be  prepared,  after  the  method  of  Diakonow, 
from  the  yolk  of  eggs.  These  are  extracted  with  ether,  then  with 
alcohol,  and  the  extracts  thus  obtained  evaporated,  when  the  result- 
ing residues  are  boiled  with  baryta  for  one  hour.  The  filtrate,  after 
the  removal  of  the  barium  by  carbonic  acid,  is  evaporated  and  the 
residue  is  extracted  with  absolute  alcohol.  The  alcoholic  solution  is 
now  precipitated  wdth  platinum  chlorid.  Brieger  (II.,  55)  has  pre- 
sented a  method  which  is  much  simpler  in  its  details  and  obviates 
the  use  of  the  expensive  platinum  chlorid.  The  tissues  rich  in 
lecithin,  as  yolk  of  eggs,  brain,  etc.,  are  heated  with  concentrated 
hydrochloric  acid  for  some  hours  on  the  water-bath.  The  insoluble 
residue  is  filtered  off,  and  the  filtrate,  after  neutralization  of  the  ex- 


296  CHEMISTRY  OF  THE  PTOMAINS. 

cess  of  free  acid  with  carbonate  of  sodium,  is  evaporated.  The 
residue  is  extracted  with  alcohol,  and  the  alcoholic  solution  is  pre- 
cipitated with  alcoholic  mercuric  chlorid.  The  precipitate  thus  ob- 
tained, on  recrystallization  several  times  from  a  large  quantity  of 
boiling  water,  yields  the  pure  double  salt  of  cholin. 

If  desirable,  it  can  be  made  from  pure  lecithin,  best  prepared  ac- 
cording to  Gilson's  method.  Yolk  of  eggs  is  repeatedly  shaken  up 
with  ether  until  the  latter  is  colored  only  a  faint  yellow ;  the  ether 
solutioji  then  distilled,  the  residue  taken  up  in  petroleum  ether  and 
filtered.  The  filtrate,  in  a  separatory  funnel,  is  well  shaken  with  75 
per  cent,  alcohol,  and  this  is  repeated  several  times  with  fresh  alco- 
hol. The  alcoholic  extracts  are  combined,  allowed  to  stand  for  some 
time,  then  filtered  and  subjected  to  distillation  to  remove  traces  of 
petroleum  ether.  The  solution  is  now  set  aside  in  a  cool  place  for 
several  days  ;  the  precipitate  which  forms  consists  of  cholesterin,  etc., 
and  a  little  lecithin.  The  alcoholic  solution  is  filtered  by  decanta- 
tion,  then  decolored  by  boiling  with  bone-black  and  rapidly  evaporated 
at  50°- 60°  to  a  syrupy  consistency.  This  residue  is  extracted  with 
ether,  the  solution  filtered  and  evaporated.  The  lecithin  thus  ob- 
tained is  almost  perfectly  pure  but  contains  traces  of  cholesterin. 
To  purify  it  completely,  it  can  be  dissolved  in  as  little  absolute 
alcohol  as  possible  and  set  aside  to  precipitate  in  the  cold  (5°-15°). 

Cholin  may  be  prepared  synthetically  according  to  the  method  of 
"Wurtz  (see  Gulewitsch,  Zeitsohr.  f.  physiol.  Chem.,  24,  514)  or  of 
Hofmann  and  Bode.  In  the  latter  ethylene  bromid  is  heated  with 
excess  of  alcoholic  trimethylamin.  The  resulting  bromin  compound 
is  treated  with  silver  nitrate,  filtered,  and  the  filtrate  heated  on  the 
water-bath  for  about  eight  days  yields  cholin  nitrate.  This  is  the 
easiest  and  cheapest  method  of  preparation.  It  may  be  prepared 
from  neurin  (p.  290). 

In  regard  to  the  genesis  of  cholin  the  preponderance  of  testimony 
goes  to  show  that  it  is  derived  from  the  decomposition  of  lecithin 
which,  according  to  the  researches  of  Diakonow  and  others,  is  one 
of  the  most  widely  distributed  compounds  occurring  in  greater  or 
less  quantity  in  all  of  the  Jtnimal  tissues.  Lecithin  which  is  a  com- 
plex ester,  decomposes  under  the  action  of  acids  and  alkalis  into  a 
base  (cholin),  glycerin,  phosphoric  acid  and  fatty  acids  (stearic,  oleic, 
palmitic,  etc.).  Gilson  has  shown  that  dilute  sulphuric  acid  slowly 
decomposes  lecithin,  forming  cholin  which,  after  a  few  days,  disap- 
pears ;  on  the  other  hand  sodium  hydrate,  in  even  1  per  cent,  solu- 
tion, rapidly  decomposes  it.  This  change  is  undoubtedly  accom- 
plished in  a  similar  manner  through  the  agency  of  bacteria.  Brieger 
(II.,  17)  is  inclined  to  believe  that  cholin  exists  preformed  in  the 
various  tissues  inasmuch  as  he  has  been  unable  to  obtain  it  from  the 
brain,  which  is  rich  in  lecithin,  by  boiling  with  2  per  cent,  hydro- 
chloric acid.     (See    Schulze,  page    298.)     Prolonged  heating  with 


CHOLIN.  297 

concentrated  hydrochloric  acid  was  necessary  in  order  to  obtain  any 
cholin  from  the  brain.  Tliis  result  of  Brieger's  is  somewhat  at  var- 
iance with  that  of  Marino-Zuco  (see  Relazione,  etc.,  pages  29,  30 
and  38)  who  obtained  from  25  grams  of  lecithin,  by  the  method  of 
Stas,  a  small  quantity  of  the  aurochlorid  of  a  base,  while  from  a  sim- 
ilar amount  he  obtained  more  relevant  quantities  by  the  method  of 
Dragendorff. 

The  occurrence  of  cholin  in  the  vegetable  kingdom  would  be  in- 
explicable to  us  at  present  were  it  not  that  we  now  know  of  the  ex- 
istence of  lecithin-like  bodies  in  plants,  from  the  decomposition  of 
which  substantially  the  same  products  are  obtained  as  from  the 
lecithin  obtained  from  the  animal  tissues.  The  existence  of  such  a 
body  in  plants  was  first  predicted  by  Scheibler  in  1870,  who  was 
led  to  this  conclusion  in  his  celebrated  study  of  beet-root  sugar 
because  of  the  presence  of  oleic  acid,  glycerin,  phosphoric  acid,  and 
betain,  as  well  as  cholesterin,  in  the  beet-root  extracts.  This  hy- 
pothesis was  confirmed  by  Hoppe-Seyler,  who,  in  1879,  found  a 
lecithin-substance  in  yeast.  Schulze  found  a  similar  compound  in 
the  cotyledons  of  lupine,  while  Jacobson  observed  its  presence  in 
mustard-seeds,  in  fenugreek-seeds,  in  maize  and  wheat,  in  the  fat 
from  beans,  peas,  vetch,  and  lupines.  Heckel  showed  its  presence 
in  globularia,  and  Lippmann  has  found  it  in  beet-root.  According 
to  Hoppe-Seyler,  this  lecithin-like  substance  exists  in  all  vegetable 
cells  undergoing  development.  Schulze  and  Likiernik  (1891)  were 
the  first  to  prepare  lecithin  in  a  pure  condition  from  plants.  It 
was  found  to  possess  the  same  properties  and  yield  the  same  decom- 
position products  as  lecithin  from  animal  tissues.  Up  to  the  present 
time  lecithin  has  always  been  supposed  to  contain,  as  an  essential 
component,  a  radical  which  gives  rise  to  cholin  on  saponification, 
while  on  the  other  hand  the  fatty  acids  entering  its  molecule  are 
well  known  to  be  replaceable  by  one  another.  Thus  we  may  have 
a  di-stearin  lecithin  as  well  as  di-olein  lecithin.  The  existence  of 
several  lecithins  in  the  yolk  of  eggs  has  been  recognized  for  some 
time,  and  according  to  Schulze  and  Likiernik  this  is  also  true  of  the 
lecithins  in  plants.  Recent  observations  of  Lippmann  {Ber.,  20, 
3206)  show  that  the  above  basic  radical,  hitherto  regarded  as  con- 
stant in  lecithin,  may  possibly  be  capable  of  replacement  by  other 
similar  radicals.  He  found  on  saponifying  with  baryta  two  different 
specimens  of  lecithin,  both  obtained  from  beet-root,  that  while  one  of 
them  yielded  oleic  acid,  glycerin,  phosphoric  acid,  and  betain,  the 
other  lecithin  gave  oleic  acid  (and  some  other  fatty  acids),  glycerin, 
phosphoric  acid,  and  cholin,  with  no  betain — at  least  not  in  isolable 
quantity.  This  remarkable  difference  has  led  Lippmann  to  suggest 
an  explanation  which,  while  it  may  not  be  the  correct  one,  neverthe- 
less possesses  a  high  degree  of  probability.  According  to  him,  the 
lecithin  molecule  may  contain  interchangeable  basic  radicals  in  the 


298  CHEMISTRY  OF  THE  PTOMAINS. 

same  manner  that  it  contains  interchangeable  acid  radicals.     This 

view  is  supported  not  only  in  the  case  of  beet-root,  where  cholin  and 

betain  exist  together,  but  the  same  two  bases  have  been  observed  in 

cotton-seed.     A*  similar  coexistence  was  observed  in  the  toad-stool 

(Agaricus   muscarius),  in   which  cholin  and  muscarin  were  found. 

And,  lastly,  the  same  condition  holds  true  probably  for  mytilotoxin 

and  betain,  which  were  shown  to  be  present  together  in  poisonous 

mussels. 

Lecithin  cannot  always  be  regarded  as  the  source  of  cholin  in 

plants,  since  this  base  is  known  to  occur  as  a  glucosid  in  the  seeds 

of  white  mustard.     The  sinapin  decomposes  according  to  the  equa- 

tioH  : 

Ci,H,3N05  +  2H,0  =  C5H15NO,  +  CiiHi^Os. 
Sinapin.  Cholin.       Sinapic  Acid. 

According  to  Schulze  (1891),  the  cholin  which  is  isolated  from 
pea-  and  vetch-seeds  exists  preformed  in  the  seeds  and  does  not  re- 
sult from  lecithin  by  the  process  of  extraction.  This  is  also  proba- 
bly true  with  reference  to  cotton-seed  cake.  The  condition  in  which 
betain  exists  is  not  determined. 

Decompositions  of  Cholin. — Baeyer  (1866)  succeeded  in  con- 
verting cholin  into  neurin  by  a  purely  chemical  process.  This  was 
accomplished  by  heating  cholin  chlorid  with  concentrated  hydriodic 
acid  and  red  phosphorus  in  a  sealed  tube  at  120°- 150°,  whereby  the 
compound  C^HjgNIg  was  formed.  Fuming  hydrobromic  acid  heated 
to  160°- 170°  may  also  be  employed.  The  iod-iodid  of  cholin  thus 
obtained,  on  treatment  with  moist  silver  oxid  (page  290),  gave  a 
base,  the  platinochlorid  of  which  corresponded  to  the  formula 
(C5Hj2NCl)2PtCl^  -t-  up.  This  double  salt,  according  to  Baeyer, 
is  readily  soluble  in  water  and  gives  reactions  similar  to  cholin. 
Although  Baeyer  is  emphatic  in  his  assertion  that  this  is  the  vinyl 
compound  (neurin)  formed  from  the  oxyethyl  base  (cholin),  yet  it 
seems  that  there  is  room  for  doubt  in  regard  to  the  interpretation 
of  his  results.  Thus  neurin  platinochlorid  is  difficultly  soluble  in 
water,  contrary  to  the  behavior  of  the  platinochlorid  obtained  by 
him.  On  the  other  hand,  cholin  platinochlorid  is  easily  soluble  in 
water,  and  it  would  seem,  therefore,  that  Baeyer  has  not  converted 
cholin  into  neurin,  but  rather  has  regenerated  cholin  from  its  iod- 
iodid.  If  such  were  the  case,  we  would  expect  that  the  iod-iodid  of 
neurin,  C^HjgNIj,  which  has  the  same  composition  as  the  corre- 
sponding derivative  of  cholin,  would  yield,  on  treatment  with 
silver  oxid,  the  oxyethyl  base.  Baeyer  was  apparently  not  able  to 
effect  this  change,  since  he  held  that  the  vinyl  base  may  be  pre- 
pared from  the  oxyethyl,  but  that  the  reverse,  the  preparation  of 
the  oxyethyl  base  from  the  vinyl  compound,  could  not  be  brought 
about.       This,    however,    has    been   successfully    accomplished   by 


CHOLIN.  299 

Schmidt.  Neurin  can  be  changed  into  cholin,  and  vice  versa  cholin 
can  be  changed  into  neurin  (page  290). 

Whether  the  change  described  by  Baeyer  takes  place  or  not,  it  is 
nevertheless  certain  that  cholin  does  not  readily  give  up  a  molecule 
of  water,  and  thus  become  converted  into  neurin.  Ch.  Gram  an- 
nounced, in  1886,  that  cholin  chlorid  and  lactate,  on  heating  on  the 
water-bath  with  dilute  hydrochloric  acid,  decompose  and  that  this 
conversion  into  the  vinyl  base  was  easy  and  complete  when  the  aque- 
ous hydrochloric  acid  solution  of  cholin  platinochlorid  was  heated 
for  five  or  six  hours  on  the  water-bath.  In  this  way  Gram  endeav- 
ored to  explain  the  formation  of  neurin  as  due  to  the  action  of  acids 
upon  cholin,  but  Brieger  has  shown  that  the  platinum  salt  of  cholin, 
as  w^ell  as  its  hydrochlorid,  can  be  heated  with  fifteen  or  thirty  per 
cent.,  or  even  concentrated  hydrochloric  acid  for  six  or  eight  hours 
without  undergoing  any  change  into  neurin,  thus  disproving  the  re- 
sults obtained  by  Gram.  E.  Schmidt  and  Weiss  have  independently 
confirmed  Brieger's  observations  in  regard  to  the  resistance  of  cholin 
to  decomposition  by  acids.  Schmidt  has  gone  further,  and  has 
shown  by  an  examination  of  Gram's  original  preparations  that  it  was 
cholin  and  not  neurin.  Gulewitsch  (1894)  was  likewise  unable  to 
split  up  cholin  by  acids  into  neurin.  What  the  action  of  acids  has 
failed  to  do  is  probably  accomplished  through  the  agency  of  bacteria. 
Schmidt  found  that  cholin  chlorid,  when  allowed  to  stand  with  hay 
infusion,  or  with  dilute  blood,  for  fourteen  days  at  20°— 30°,  decom- 
posed almost  entirely,  yielding  large  quantities  of  trimethylamin  and 
a  base,  the  platinochlorid  of  which  resembles  in  form  and  solubility 
the  double  salt  of  neurin  and  possesses  a  similar  physiological  action. 
When  allowed  to  decompose  for  ten  days  at  30°- 33°  neither  cholin 
nor  neurin  was  present.  Cholin  lactate  in  hay  infusion  developed  an 
odor  of  trimethylamin  in  twelve  hours,  but  at  the  end  of  fourteen 
days  a.good  deal  of  cholin  was  still  present.  In  this  case  no  neurin 
was  present,  but  instead  a  homologous  base  was  found  which  can  be 
obtained  synthetically  by  the  action  of  trimethylamin  on  allyl  bro- 
mid.  According  to  Meyer,  of  Marburg,  this  base  does  not  possess 
the  muscarin-like  action  of  neurin,  but  resembles  more  closely  pilo- 
carpin. 

The  decomposition  of  cholin  by  putrefaction  into  neurin,  and  pos- 
sibly muscarin,  highly  poisonous  bases,  may  explain  the  production 
of  poisons  in  foods.  Nesbitt  has  in  like  manner  endeavored  to  show 
that  intestinal  auto-intoxication  may  be  due  to  the  formation  of  neu- 
rin out  of  the  cholin  derived  from  the  food  lecithin  (p.  291).  The 
similar  view  that  mental  disorders  might  be  due  to  neurin  formation 
in  the  brain  led  Gulewitsch  (1899)  to  examine  the  leucomains  of 
perfectly  fresh  brain.  He  obtained  cholin,  two  bases  (possibly  di- 
amins)  and  urea,  but  no  neurin.  Neither  was  he  able  to  confirm 
Liebreich's  view  that  protagon  gave  neurin  (p.  290). 


300  CHEMISTRY  OF  THE  PTOMAINS. 

Brieger  (I.,  59)  had  unsuccessfully  tried  to  transform  cholin  into 
neurin  by  putrefaction.  He  observed  that  the  cholin  decomposed 
with  extreme  slowness,  even  when  the  putrefaction  was  carried  on  at 
a  higher  temperature,  yielding  only  trimethylamin.  Wurtz  (1868) 
showed  that  dilute  solutions  of  free  cholin  can  be  heated  to  boiling 
without  any  perceptible  decomposition.  Concentrated  solutions, 
however,  decompose  with  the  formation  of  trimethylamin  and  glycol, 
C2H^(OH)2  (see  page  250).  The  decomposition  of  cholin  was  studied 
somewhat  by  Mauthner  (1873)  who  confirmed  Wurtz's  observation 
that  cholin  was  scarcely  decomposed  by  boiling  water,  and  he  showed 
that  when  exposed  to  the  action  of  decomposing  blood  it  yielded 
trimethylamin.  The  results  obtained  by  K.  Hasebroek  (ZeitsGhrift 
f,  physiol.  Chem.,  12,  151,  1888)  deserve  special  mention  at  this 
place.  He  carried  on  the  putrefaction  of  very  dilute  solutions  of  the 
chlorid  of  cholin  in  the  presence  of  little  or  no  oxygen  in  Hoppe- 
Seyler  fermentation  flasks.  Sewer  slime,  because  of  its  strong  fer- 
mentative properties,  was  used  to  induce  the  putrefaction,  and  cal- 
cium carbonate  was  added  to  neutralize  any  acidity  that  might 
develop  during  the  fermentation. 

The  fermentation,  as  shown  by  the  evolution  of  gases,  lasted  for 
about  three  months.  The  total  quantity  of  gas  given  off  was  about 
one  liter  from  1.17  grams  cholin  chlorid.  The  gases  consisted  almost 
entirely  of  carbonic  acid  and  marsh  gas.  No  hydrogen  was  evolved. 
When  the  fermentation  ceased  the  flask  was  opened  and  several  cubic 
centimeters  of  the  almost  neutral  clear  liquid  were  injected  under 
the  skin  of  a  rabbit  without  producing  the  least  effect. 

This  liquid  distilled  with  alkali  gave  methylamin  and  ammonia. 
What  is  remarkable  about  this  experiment  was  the  total  absence 
of  the  higher  amins — as,  for  instance,  trimethylamin,  which  has  been 
observed  so  many  times  as  a  decomposition  product  of  cholin.  The 
absence  of  any  poisonous  base,  as  neurin,  was  probably  largely 
connected  with  the  absence  of  oxygen. 

Free  cholin  ordinarily  forms  a  strongly  alkaline  syrup  which  com- 
bines readily  with  acids  to  form  salts,  most  of  which  are  deliquescent. 
By  oxidation  it  is  converted  into  betain  (see  page  305),  and  on  treat- 
ment with  concentrated  nitric  acid  it  gives  rise  to  a  muscarin  (see 
page  309).     These  reactions  can  be  represented  by  the  equations  : 

CHjOH  CO.  OH 


? 


N(CH3)3.0H  N(CH3)3.0H 

Cholin.  Betain. 


jOH 
+  O  =     CHOH 


CH,' 


f 

N(CH,)3.0H. 

MUSCAEIN. 


CHOLIN.  301 

By  the  action  of  dilute  nitric  acid  cholin  is  converted  almost  wholly 
into  a  base  the  platinochlorid  of  which  is  efflorescent,  and  forms 
large,  bright  crystals  grouped  in  bunches.  It  corresponds  to  the 
formula  (C,Hj,N203Cl)PtCl,  +  2H,0  (Schmiedeberg  and  Harnack). 

Nothnagel  isolated  the  same  substance,  but  on  attempting  to  con- 
vert the  platinum  salt  into  the  gold  compound,  he  obtained  the 
aurochlorid  of  trimethylamin.  The  nature  of  this  base,  which  is 
formed  also  in  small  quantity  on  oxidation  with  concentrated  nitric 
acid,  is  uncertain. 

According  to  Mauthner,  cholin  resembles  the  caustic  alkalis  in  its 
action.  Although  putrefying  blood  decomposes  it  into  trimethyl- 
amin, yet,  when  present  in  the  proportion  of  1.4  per  cent.,  it  is  said 
to  arrest  putrefaction.  A  1  to  2  per  cent,  solution  like  strong  alkali 
dissolves  fibrin  or  coagulated  albumin  on  boiling. 

Nothnagel  has  shown,  contrary  to  Arndt,  that  cholin  cannot  be 
distilled  unchanged  with  baryta  water.  It  is  decomposed  into  tri- 
methylamin. On  distillation  with  water  it  yields  a  few  drops  of  an 
aldehyde  body,  a  little  neurin  (?),  and  trimethylamin.  On  dry  dis- 
tillation of  cholin  it  yields  also  a  little  of  the  aldehyde  body,  a  little 
neurin  (?),  and  chiefly  cholin.  The  latter  probably  results  from  re- 
combination in  the  distillate  of  the  trimethylamin,  ethylene  oxid, 
and  water.  In  general  it  may  be  said  that  concentrated,  but  not  di- 
lute, solutions  are  decomposed  by  boiling. 

The  free  base  forms  a  syrupy  liquid  which  eagerly  takes  up  car- 
bonic acid  from  the  air  and  is  converted  into  the  carbonate  which 
forms  elongated  six-sided  plates.  The  base  is  easily  soluble  in  water 
and  in  absolute  alcohol  but  is  insoluble  in  chloroform  or  in  ether 
(Gulewitsch). 

The  chlorid,  CgHj^NO.Cl,  is  easily  soluble  in  water  and  in  abso- 
lute alcohol  (separation  from  ueuridin  hydrochlorid  and  from  betain). 
It  is  insoluble  in  ether,  chloroform  and  benzol.  From  alkaline 
solution  traces  are  dissolved  by  amyl  alcohol.  It  crystallizes  over 
sulphuric  acid  to  needles  which  readily  deliquesce  in  the  air.  Potas- 
sium mercuric  iodid  produces  in  solution  of  the  chlorid  a  crystalline 
precipitate.  For  the  behavior  to  other  reagents  see  Table  I  (also 
Gulewitsch).     The  nitrate  possesses  the  formula  : 

(CH3)3.N(NOj).CHj.CH,OH.  (Schmidt). 

The  bromid,  Br.N(CH3)3.C2H^OH,  forms  rather  long,  colorless 
rhombic  plates  when  ether  is  added  to  an  absolute  alcohol  solution 
of  the  salt.  It  deliquesces  very  rapidly  in  the  air  and  is  decomposed 
by  sunlight,  changing  color  to  violet  and  brown  (Nothnagel). 

The  iodid,  I.N(CH3)3.C2lI^OH,  can  be  crystallized  in  the  same 
way  as  the  bromid.  It  is  less  deliquescent  and  is  turned  yellow  by 
sunlight  (Nothnagel). 

The  platinochlorid,  (C,Hj^N0.Cl)2PtCl,  (Pt  =  31.64  per  cent.). 


302  CHEMISTRY   OF  THE  PTOMAINS. 

presents  an  interesting  case  of  trimorphism.  It  crystallizes  in  mono- 
clinic  plates  (Rinne)  which  are  easily  soluble  in  water  (1  to  5.82 
parts  at  21°,  G.)  insoluble  in  alcohol,  ether,  chloroform,  benzol;  also 
in  characteristic  superposed  plates,  sometimes  in  the  form  of  orange- 
red  flat  prisms  (Brieger).  From  a  warm  saturated  solution  contain- 
ing 15  per  cent,  alcohol  it  crystallizes  in  yellow  regular  octahedra 
containing  one  molecule  of  water  of  crystallization,  from  aqueous 
solutions  in  six-sided  plates  (Jahns) ;  from  aqueous  solution  on  slow 
evaporation  it  forms  plates,  clinorhombic  plates,  or  needles  (Hoppe- 
Seyler)  which  are  anhydrous.  When  rapidly  crystallized  it  forms 
prisms  (Hundeshagen,  Jahns,  Schulze) ;  and  if  the  solution  is  con- 
centrated the  prisms  are  very  thin,  almost  needles.  According  to 
Schulze,  it  sometimes  forms  beautiful  orange-red,  chiefly  six-sided 
plates.  It  is  easily  soluble,  forming  splendid,  very  large,  red,  super- 
posed monoclinic  plates ;  may  crystallize  from  cold  saturated  aqueous 
solution  in  large,  prismatic,  or  needle-shaped  crystals  (Schmidt). 
Jahns  maintained  that  the  plates  and  prisms  belong  to  the  same 
system ;  while  Hundeshagen  held  that  they  are  distinct.  Instead 
of  the  salt  presenting  an  instance  of  trimorphism  as  first  stated  by 
Hundeshagen,  it  would  seem  that  but  two  forms  occur — anhydrous 
monoclinic  and  octahedra  with  one  molecule  of  water  of  crystalliza- 
tion. It  contains  always  more  or  less  water  of  crystallization,  which 
it  does  not  give  up  completely  over  sulphuric  acid,  but  only  at  110° 
(Brieger).  The  natural  platinochlorid  becomes  strongly  electric  on 
rubbing,  whereas  the  synthetic  cholin  double  salt  does  not  become 
electric.  It  melts  at  225°  with  effervescence  (Jahns) ;  at  about  238° 
(Partheil);  at  233°- 234°  (Bode) ;  at  232°-233°,  and  more  often 
at  240°- 241°  (Schmidt),  with  marked  effervescence.  The  synthetic 
salt  melts  at  213°- 216°  (Gulewitsch).  The  solubility,  crystalline 
form  and  melting-point  render  it  wholly  different  from  neurin. 
The  properties  of  this  and  other  salts  of  cholin  have  been  carefully 
reexamined  by  Gulewitsch  (1898). 

The  aurochlorid,  CgHj^NO.Cl.AuClj  (Au  =  44.48  per  cent.),  is 
crystalline  and  is  difficultly  soluble  in  cold  water,  but  can  be  recrys- 
tallized  from  hot  water  or  from  boiling  alcohol.  It  forms  prisms, 
or  gold-yellow  long  needles  which  are  very  easily  soluble  in  hot 
water  and  alcohol  (Lippmann).  It  may  form  cub6s  and  at  23.5°  it 
dissolves  in  67.2  parts  of  water  (G.).  It  can  be  separated  from 
neuridin  aurochlorid  by  its  solubility  in  water  (Brieger).  On  heat- 
ing the  gold  salt  melts  to  a  brown  liquid  (Schulze),  and  decomposes 
at  264°.  It  melts  at  244°- 245°  (Siebert,  Jahns) ;  at  245°-246° 
(Schmidt)  ;  at  241°-243°  (G.). 

The  picrate,  C5Hj^NO.OC5H2(N02)3,  forms  long,  broad  needles 
which  are  more  easily  soluble  than  neuridin  picrate,  and  hence  can 
be  separated  by  recrystallization.  It  is  more  easily  soluble  in  alco- 
hol than  in  water. 


CUOLIN.  303 

The  raercurochlorid,  CgHj^NO.Cl  +  eHgClg,  is  difficultly  soluble 
even  in  hot  water.  On  this  account  the  mercury  salt  is  very  con- 
venient for  the  separation  of  cholin  from  accompanying  bases.  It 
is  very  difficult  to  separate  cholin  from  cadaverin  mercurochlorid 
on  account  of  the  similarity  in  solubilities.  Gulewitsch  secured  a 
separation  by  fractional  precipitation  and  fractional  solution.  It 
forms  small,  short,  cross-shaped  prisms.  It  melts  at  249°- 251° 
(G.)  ;.  at  242°  (Morner).  Its  solubility  in  water  is  1  :  56.6  at  24.5° 
(Gulewitsch);  at  1  :66.6,  19.5°  (Morner).  Like  creatinin  it  yields 
a  crystalline  precipitate  with  alcoholic  zinc  chlorid  (G.). 

Lactocholin,  gif gi^jjigijiggfcH.O.Oc)  CH-CH.     This  compound 

was  obtained  by  Schmidt  by  heating  an  aqueous  solution  of  cholin 
lactate  on  a  water-bath  for  six  days.  The  platinochlorid  forms  long 
prismatic  crystals  with  roof-shaped  ends.  It  is  easily  soluble  in 
water ;  difficultly  in  alcohol.  It  has  two  molecules  of  water  of 
crystallization,  and  melts  at  220°— 221°.  The  platinum  compound 
on  decomposition  with  hydrogen  sulphid  or  potassium  chlorid  and 
precipitation  with  gold  chlorid  yields,  instead  of  a  gold  salt,  the 
aurochlorid  of  cholin.  All  attempts  to  obtain  the  aurochlorid  of 
lactocholin  failed.  Lactocholin  is  formed  from  ethylidene,  and  not 
from  ethylene  lactic  acid  (Nothnagel). 

Oxy-iso-butyro-cholin  is  produced  in  the  same  way  as  lactocholin. 
The  platinochlorid  has  the  same  form  and  melting-point — 221°. 
The  two  molecules  of  water  of  crystallization  are  difficultly  driven 
off.  The  gold  salt  is  not  obtainable  on  account  of  reversion  to 
cholin.  The  formula  corresponds  to  that  of  lactocholin  (Noth- 
nagel). 

Oxy-valero-cholin  is  also  prepared  the  same  as  lactocholin.  The 
platinum  salt  crystallizes  with  two  molecules  of  water  of  crystalliza- 
tion, in  long  compact  needles,  which  are  rather  easily  soluble  in 
water,  and  melt  at  223°- 224°  (Nothnagel).  Its  composition  also 
corresponds  to  that  of  lactocholin. 

Oxy-acetic,  ethylene  lactic,  and  salicylic  acids  do  not  form  anhy- 
drid  compounds.  The  above  bodies  result  from  the  union  of  two 
molecules  of  cholin  and  one  molecule  of  acid*  with  elimination 
of  two  molecules  of  water.  The  platinum  salts  of  all  three  anhy- 
drides belong  to  the  same  system ;  all  have  two  molecules  of  water 
of  crystallization,  difficultly  expelled  at  100°.  The  melting-point 
is  about  the  same  in  all.  They  do  not  yield  gold  salts.  The  free 
anhydrids  are  not  permanent. 

Acetyl  cholin.  The  gold  salt  of  this  compound  was  studied  first 
by  Baeyer,  later  by  Nothnagel.  Baeyer  obtained  the  acetyl  com- 
pound by  the  action  of  acetyl  chlorid  on  cholin  chlorid  in  the  cold, 
but  Nothnagel  did  not  succeed  in  introducing  the  acetyl  group  short 


304  CHEMISTRY  OF  THE  PTOMAINS. 

of  100°.  The  gold  salt  is  anhydrous,  dendritic  in  form,  and  melts 
at  154°- 155°.  On  decomposition  with  hydrogen  sulphid  it  yields 
cholin.  The  platinochlorid  crystallizes  in  small  anhydrous  needles 
which  melt  at  223°- 224°. 

Benzoyl  cholin  is  formed  by  heating  dry  cholin  chlorid  on  a  water- 
bath  with  benzoyl  chlorid.  It  forms  a  platinum  salt  crystallizing 
in  fine  thread-like  needles  which  melt  at  206°.  The  gold  salt 
forms  light-yellow  flat  needles  which  are  permanent  in  the  air 
and  melt  at  183°.  The  hydrogen  in  the  hydroxyl  group  of 
the  oxy ethyl  is,  therefore,  easily  replaced  by  acid  radicals  (Noth- 
nagel). 

jPhysiologkal  Action  of  Cholin. — Cholin  was  regarded  for  a  long 
time  as  physiologically  inert,  but  this  belief  was  set  aside  by  Gaeht- 
gens  (1870),  who  showed  that,  when  given  in  large  quantity,  it  pos- 
sessed a  marked  toxic  action  ;  0.59  gram  producing  almost  instan- 
taneous death  in  a  cat.  This  observation  of  Gaehtgens  has  since 
been  confirmed  by  Glause  and  Luchsinger,  Brieger,  and  Boehm. 
The  chlorid  of  cholin  produces  in  animals  the  same  muscarin-like 
symptoms  of  poisoning  as  are  developed  by  the  vinyl  base  neurin,  the 
only  difference  lies  in  the  intensity  of  the  action.  In  order  to  bring 
about  a  physiological  disturbance,  cholin  must  be  given  in  relatively 
large  doses.  Thus  Brieger  found  it  necessary  to  give  about  0.1 
gram  of  cholin  chlorid  hypodermically  to  a  one  kilogram  rabbit  in 
order  to  bring  out  the  same  effects  as  are  obtained  by  the  injection 
of  0.005  gram  of  the  neurin  salt.  He  also  found  that  the  fatal  dose 
for  a  one  kilogram  rabbit  was  about  0.5  gram,  which  is  about  ten 
times  as  large  as  the  fatal  dose  of  neurin  chlorid.  Boehm  observed 
that  doses  of  0.025-0.1  gram  produced  in  frogs  general  paralysis, 
which,  in  a  short  time,  led  to  death  or  recovery ;  and  that  in  its 
curara-like  paralyzing  action  cholin  resembled  artificial  muscarin, 
although  the  latter  is  about  five  hundred  times  stronger.  Atropin, 
as  in  the  case  of  neurin  and  muscarin,  antagonizes  the  action  of 
cholin.  Thus,  0.05  gram  of  the  chlorid  produced  in  a  frog  in  one 
hour  diastolic  stoppage  of  the  heart.  This  condition  was  removed 
by  the  injection  of  0.001  gram  of  atropin,  the  heart-beat  rising  to 
the  normal  in  about  fourteen  minutes ;  0.05  gram  of  cholin  chlorid, 
given  subcutaneously  to  a  rabbit  (1,250  grams),  produced  salivation, 
which  lasted  but  a  short  time  and  did  not  affect  the  heart-beat  and 
respiration ;  0.10  gram  was  necessary  to  bring  out  all  the  symptoms  ; 
0.05  gram,  given  to  guinea-pigs,  had  no  effect  whatever.  Accord- 
ing to  Halliburton  (1901)  it  causes  a  dilatation  of  the  peripheral 
blood  vessels,  especially  those  of  the  intestines,  and  hence  a  fall  of 
blood  pressure. 

Betain  (oxyneurin),  CgHjgNOg. — This  base  has  been  well  known 
for  some  time,  because  of  its  occurrence  in  the  vegetable  kingdom. 


BETAIN.  305 

Thus,  it  is  present  in  cotton-seed  (Boehm,  Ritthausen  and  Weger, 
Maxwell),  where  it  is  about  five  times  as  abundant  as  cholin ;  in  beet- 
root juice  (Beta  vulgaris),  and  hence  in  beet-root  molasses  (Scheibler, 
1866).  It  occurs  also  in  cattle-turnip  and  Lycium  barbarum  (Huse- 
mann  and  Marm^,  Schiitte,  Siebert),  and  is  found  with  cholin  and 
another  base  in  vetch-seeds ;  in  peas  a  base  similar  to  betain  exists 
(Schulze).  With  cholin  it  occurs  in  the  roots  and  leaves  of  Scopolia 
atropoides  (Siebert).  It  occurs  in  the  leaves  of  the  potato  plant,  So- 
lanum  tuberosum,  but  not  with  cholin  (Schiitte) ;  in  worm-seed  (Arte- 
misia Cina)  in  about  0.5  per  cent.;  with  cholin  about  0.1  per  cent. 
(Jahns).  The  two  bases  are  also  present  in  the  sprouts  of  wheat  and 
malt,  betain  more  abundantly  (Schulze  and  Frankfurt).  It  does  not 
exist  in  these  substances  as  such,  but  is  formed  from  a  more  complex 
substance  by  the  action  of  hydrochloric  acid  or  baryta  (Liebreich). 
In  this  respect  it  resembles  cholin,  neurin  and  probably  muscarin. 
Quite  recently,  Lippmann  (1887)  has  obtained  a  lecithin-like  body 
from  sugar-beet,  which,  on  heating  with  baryta,  gave  oleic  acid,  gly- 
cerin, and  phosphoric  acid  (glycerin-phosphoric  acid),  and  betain. 
Betain,  however,  does  not  seem  to  be  a  constant  constituent,  inasmuch 
as  on  one  occasion  he  obtained  chiefly  cholin,  and  little  or  no  betain. 
These  two  bases  also  occur  together  in  cotton-seed,  and  this  fact  has 
led  Scheibler  to  the  conclusion  that  it  is  no  mere  chance.  Lecithin,  as 
is  well  known,  may  contain  variable  acid  constituents  (oleic,  stearic, 
palmitic,  etc.),  and  reasoning  on  this  fact,  and  on  the  results  of  his 
own  experiments,  Lippmann  was  led  to  suppose  that  it  may  also 
contain  different  bases  in  variable  proportions. 

Betain  was  first  discovered  by  Husemann  and  Marme  in  1863  and 
1864  and  named  lycin.  Scheibler  found  it  in  1866  in  beets  and 
gave  it  the  present  name.  The  identity  of  the  two  compounds  was 
shown  in  1875  by  Husemann. 

A  methyl  betain,  trigonellin,  exists  in  trigonella  (Jahns,  Hantzch). 

It  has  been  obtained  from  human  urine  (Liebreich,  1869),  and 
from  poisonous  and  non-poisonous  mussel,  but  not  from  putrid 
mussel  (Brieger,  1885,  III.,  76).  Emmerling  (1896)  obtained 
it  with  trimethylamin  by  decomposing  gluten  with  proteus  vul- 
garis. The  method  for  its  separation  from  mussel  is  described  on 
page  313. 

Betain  may  be  obtained  synthetically  in  several  ways :  (1)  By 
oxidation  of  cholin  with  potassium  permanganate ;  (2)  by  heating 
sarkosin  (methyl  glycocoll)  with  methyl  iodid  and  methyl  alcohol,  or 
with  methyl  iodid  alone,  when  betain-methyl  ether  also  forms  (Paul- 
mann) ;  (3)  by  the  action  of  silver  oxid  on  betain  aldehyde ;  (4)  by 
the  action  of  methyl  iodid  on  glycocoll  (Kraut)  ;  (5)  by  treating 
monochloracetic  acid  with  trimethylamin.  The  last  two  methods 
are  of  value  as  indicating  the  constitution  of  betain,  and  the  changes 
which  take  place  can  be  represented  by  the  equations  : 
20 


306  CHEMISTRY  OF  THE  PTOMAINS. 

NH,  I^(CHs),I 

JHj  +  3CH3I  =  CHj  +  2HL 


CHj  +  3CH3I  =  CI 


CO^H  CO2H 

Glycocoll.  Betain  Iodid. 

CH.C1  ^*^='>'''' 


l^j^jj    +N(CH.),=  ^H, 

C02H. 

MONOCHLOR- 

ACETic  Acid. 

Another  method  of  synthesizing  anhydrous  betain  consists  in  heat- 
ing di-methyl  amido-acetic  methyl  ester.  The  reaction  is  reversible 
(Wildstatter,  Ber.,  35,  585,  597). 

From  the  formulae  of  the  salts  of  betain  it  is  evident  that  betain 
has  properly  the  composition  CgH^jNOg  which  is  expressed  by  the 
structural  formula : 

N(CH3)30H. 


CO2H. 

The  free  base  is,  however,  readily  converted  into  the  anhydrid, 
CgH^jNOg,  trimethyl  glycocoll,  the  structural  formula  of  which  is  : 

H,-N(CH3), 
0  —  0. 

Betain  aldehyde  was  prepared  first  by  Berlinerblau  and  later  by 
Fischer.     On  oxidation  with  silver  oxid  it  yields  betain. 

Betain  is  ordinarily  regarded  as  crystallizing  with  one  molecule  of 
water,  and  the  composition  is  expressed  by  the  formula  :  CgHj^NOj 
+  'S.p{=  OH.N(CH3)3.CH2.C02H).  It  loses  this  water  of  crystal- 
lization by  heating  at  100°,  or  on  standing  over  sulphuric  acid, 
forming  an  anhydrid  of  the  formula  already  given.  The  anhydrid 
is  very  hygroscopic,  and  melts  at  293°  (Wildstatter).  Liebreich 
claimed  that  free  betain  possessed  the  formula  CgHj^NOg  because  it 
yielded  a  compound  having  the  composition  (CgHjjNO)ZnCl2.  The 
free  base  separates  from  alcohol  in  large  crystals  which  deliquesce  on 
exposure  to  the  air.  As  obtained  by  Brieger  from  the  hydrochlorid, 
by  treatment  with  moist  silver  oxid,  it  possessed  a  sweetish  taste  and 
neutral  reaction.  When  distilled  with  potassium  hydrate,  it  yielded 
trimethylarain  and  other  bases,  among  which  a  base  of  the  formula 
CgHjyNOj  occurred  in  the  largest  quantity.  In  1893  Scheibler  again 
studied  the  action  of  sodium  hydrate  on  betain  and  found  only  tri- 
methylarain ;  no  new  base,  but  unchanged  betain. 

The  chlorid,  C^Hj2N02.Cl,  forms  beautiful  monoclinic  plates 
which  are  permanent  in  the  air,  and  this  fact  can  be  made  use  of 
to  effect  a  separation   from  the  cholin  salt,  which  is  deliquescent. 


MUSCARIN.  307 

It  ciystallizes  from  aqueous  solution  in  monoclinic  plates,  from  hot 
saturated  80  per  cent,  alcoholic  solution  in  beautiful  prisms,  often 
several  cm.  long.  It  melts  at  227°- 228°  (Jahns).  It  is  insoluble 
in  absolute  alcohol.  This  fact  can  be  made  use  of  in  their  separa- 
tion (Lippmann,  Maxwell).  It  can,  moreover,  be  easily  separated 
from  other  bases  by  its  aurochlorid  which  is  easily  soluble.  If  a 
little  potassio-mer curie  iodid  is  added  to  a  solution  of  the  chlorid, 
there  forms  a  light  yellow  or  whitish  oily  precipitate,  which  is  sol- 
uble in  excess,  but  on  rubbing  the  sides  of  the  tube  with  a  glass  rod 
it  reappears  as  yellow  needles.  This  is  said  to  be  a  characteristic 
test  (Brieger,  Schulze,  1891).  By  the  action  of  sodium  amalgam 
on  aqueous  solutions  of  the  chlorid  a  base  is  formed,  the  platino- 
chlorid  of  which  in  form,  solubility,  and  composition  agrees  with 
muscarin  (Schmidt,  Nothnagel).  An  iodid  and  a  potassium  iodid 
compound  are  known  (Wildstiitter). 

The  aurochlorid,  CjHj^NO^.Cl.AuClg  (Au  =  43.12  per  cent.), 
forms  magnificent  cholesterin-like  four-sided  plates  (or  gold  yellow 
needles,  Paulmann),  and  is  easily  soluble  (Brieger).  The  aurochlorid 
from  sugar-beet  is  said  to  crystallize  in  needles  and  plates,  and  to 
be  difficultly  soluble  in  cold  water  (Scheibler,  Lippmann).  The 
double  salt  of  the  ptomain  melts  at  209°  and  in  this  it  coincides 
with  that  obtained  from  beet-sugar,  as  well  as  with  that  of  the  syn- 
thetically prepared  base  (Brieger).  According  to  Schiitte,  it  melts 
•at  218°,  220°-222°  ;  at  223°-225°  (Siebert)  ;  at  220°- 221°,  de- 
composing at  222.5°  (Paulmann)  at  230°- 235°  (Fischer);  at  227° 
(Emmerling).  The  platinochlorid,  (C5H,^N02.HCl)2.PtCl^,  is  yellow, 
crystallizes  in  prisms  and  is  easily  soluble  (E.).  On  rapid  cooling 
of  hot  saturated  solution  or  on  precipitation  with  alcohol  it  forms 
more  or  less  anhydrous  fine  needles  ;  from  cold  saturated  solution 
over  sulphuric  acid  it  crystallizes  in  plates  which  effloresce  in  the  air 
(Jahns).  It  may  crystallize  with  or  without  water.  Liebreich  and 
Wildstiitter  obtained  crystals  with  four  molecules,  while  Paulmann 
obtained  crystals  with  one  molecule  of  water.  Jahns  obtained  the 
salt  with  three  molecules  of  water. 

Betain  is  not  poisonous.  It  is  precipitated  with  mercuric  chlorid 
together  with  cholin.  Schulze  and  Frankfurt  separate  the  mercury 
salts  of  betain  and  cholin  by  partial  crystallization  ;  betain  is  more 
soluble.  The  two  bases  can  be  separated  as  chlorids  by  the  solubil- 
ity in  absolute  alcohol  (Maxwell,  Schulze  and  Frankfurt,  Jahns). 

Muscarin,  CgHj^NOg  =  C^II^gNOg  +  Hf),  is  the  well-known 
toxic  principle  which  Schmiedeberg  and  Koppe  obtained  from  poi- 
sonous mushroom  (Agaricus  muscarius),  in  which  it  is  present  ac- 
companied by  cholin  (Harnack).  Bbhm  found  cholin  and  muscarin 
together  in  Boletus  luridus  and  Amanita  pantherina.  Later, 
Schmiedeberg  isolated    from  a  commercial    specimen  of   muscarin 


308  CHEMISTRY  OF  TEE  PTOMAINS. 

a  base  possessing  an  antagonistic  action  to  muscarin,  Kobert  be- 
lieved that  this  "  fuugus-atropin  "  existed  in  the  fresh  mushrooms, 
and  showed  that  Russula  emetica  contained  this  compound  as  well  as 
cholin  and  muscarin. 

This  base  is  especially  interesting  because  of  the  relation  it  bears 
to  cholin,  for  Schmiedeberg  and  Harnack  showed  that  it  is  formed 
when  cholin,  or,  better  still,  the  platinochlorid,  is  oxidized  by 
concentrated  nitric  acid,  Nothnagel  by  the  action  of  concentrated 
nitric  acid  on  cholin  obtained  muscarin,  also  a  nitroso  derivative 
(nitric  acid  and  cholin  ether),  and  a  substance  which  is  the  chief 
product,  besides  a  little  muscarin  and  the  nitroso  compound,  when 
the  oxidation  is  carried  out  with  dilute  nitric  acid.  The  muscarin 
from  cholin  does  not  combine  with  phenyl-hydrazin ;  betain  alde- 
hyde does.  The  chlorid  on  treatment  with  acetic  anhydrid  or  ben- 
zoyl chlorid  yields  an  anhydrid  of  muscarin  (Nothnagel),  the  exact 

composition  of  which  is  yet  undetermined  ;  the  group  — CH<('qtt  in 

muscarin  is  probably  changed  into  an  aldehyde  group  — COH. 

In  the  preparations  of  muscarin  from  cholin  a  small  quantity  of 
a  nitroso  compound  forms,  the  platinum  salt  of  which  resembles  that 
of  muscarin  in  solubility,  but  never  in  form,  which  is  always  plu- 
mose. These  crystals  are  permanent  in  the  air  and  contain  two 
molecules  of  water  which  are  not  driven  off  at  100°.  They  melt 
at  223°-  224°  with  decomposition.     It  possesses  the  formula  : 

(Cl.N(CH3)3.CH3.CH20.NO)2.PtCl^  +  2HjO. 

The  gold  salt  forms  fine,  light-yellow  needles  which  are  anhydrous 
and  melt  at  240°.  It  gives  Liebermann's  nitroso  reaction — blue 
color  with  phenol  and  sulphuric  acid. 

In  addition  to  the  natural  and  synthetic  bases  a  third  "  muscarin," 
OH.N(CH3)3.CH2.COII,  was  prepared  by  Berlinerblau  by  the  action 
of  baryta  on  trimethylamin  and  chloracetal.  Fischer  prepared  the 
same  compound,  by  the  action  of  concentrated  hydrochloric  acid  on 
acetal-trimethyl  ammonium  hydroxid.  This  base,  however,  differs 
from  muscarin  by  the  elements  of  water — anhydro-muscarin.  In 
reality  it  is  betain  aldehyde,  since  on  oxidation  with  moist  silver 
oxid  it  yields  betain  (Fischer).  Unlike  real  muscarin,  it  has  no 
action  on  the  heart  of  frogs  or  on  the  pupils  of  birds  (Meyer).  Like 
most  ammonium  bases  it  induces  strong  salivation  and  perspiration. 
Schmiedeberg  found  it  to  resemble  cholin  in  its  action,  whereas 
Luchsinger  found  it  to  agree  in  this  respect  with  muscarin. 

A   fourth   base,   oxycholin— OH.N(CH3)3.CH.OH.CHpH,    was 

prepared  by  Bode  by  the  action  of  silver  oxid  on  hypochlorous  acid 

and  neurin  chlorid.     Its  platinum  salt  melts  at  254°  (Nothnagel). 

Its  physiological  action  is  different  from  that  of  muscarin.     Thus, 

n  frogs  it  slows  the  heart,  but  does  not  cause  stoppage.     Atropin 


MUSCABIN.  309 

counteracts  its  action.  In  mammals  the  pulse  is  lowered  as  a  result 
of  stimulation  of  the  central  vagus  ganglia.  The  blood  pressure  is 
not  lowered  as  is  the  case  in  muscarin,  but  is  somewhat  raised. 
Neither  the  intestines  nor  the  iris  of  mammals  is  affected.  The  iris 
of  birds  is  contracted  as  with  muscarin,  and  the  glands  are  affected. 
In  cats  and  guinea-pigs  salivation  and  flow  of  tears  result.  Like 
all  ammonium  bases  it  has  a  marked  curara  action. 

A  fifth  base,  resembling  fungus-  and  cholin-muscarin  in  the  form, 
solubility  and  composition  of  the  platinochlorid,  was  obtained  by  the 
action  of  sodium  amalgam  on  aqueous  solutions  of  betain  chlorid 
(Schmidt). 

Lastly,  Brieger  in  1885  (I.,  48)  isolated  a  muscarin  base  from 
haddock  which  had  been  allowed  to  decompose  for  five  days.  The 
process  by  which  its  isolation  was  effected  is  described  on  page  315. 
Gulewitsch  isolated  a  small  amount  of  a  substance  resembling  mus- 
carin, together  with  cholin  and  cadaverin,  from  horse-flesh  kept  at 
15°  for  four  months. 

It  is  barely  possible  that  Brieger's  base  is  distinct  from  Schmiede- 
berg's ;  nevertheless,  it  closely  resembles  it  and  apparently  is  iden- 
tical. 

The  chlorid,  Cgll^^NOg.Cl,  is  obtained  on  the  decomposition  of  the 
platinochlorid  with  hydrogen  sulphid,  as  a  syrupy  residue  which, 
under  the  desiccator,  shows  a  tendency  to  crystallize  gradually 
(Brieger).  It  is  deliquescent  (Harnack).  A  commercial  muscarin 
sulphate  was  found  to  be  chiefly  cholin  (Nothnagel). 

The  platinochlorid,  (C5Hj,N02.Cl)2PtCl,  (Pt  =  30.08  per  cent., 
Brieger),  forms  a  crystalline  deposit  of  more  or  less  well-formed  octa- 
hedra,  pinhead  in  size,  which  are  difficultly  soluble  in  water.  They 
lose  their  water  of  crystallization  (2H2O)  only  on  strong  heating 
(Brieger,  Nothnagel).     It  melts  at  about  240°  with  decomposition. 

The  aurochlorid,  C.Hj.NO^.Cl.AuClg  (Au  =  42.82  per  cent.), 
crystallizes  in  needles,  and  is  difficultly  soluble  in  water  (Brieger)  ; 
more  difficultly  soluble  than  the  cholin  double  salt  (Harnack). 
From  hot  hydrochloric  acid  water  it  crystallizes  as  light-yellow, 
glistening  platelets  (Nothnagel).  It  is  scarcely  to  be  distinguished 
from  the  corresponding  salt  of  cholin  (Nothnagel).  It  begins  to  run 
together  at  174°,  gradually  melts  and  decomposes  at  232°.  It 
has  no  water  of  crystallization.  The  separation  of  muscarin  and 
cholin  is  very  difficult.  Harnack  separated  the  two  by  spreading 
the  mixed  chlorids  on  a  filter  paper  which  absorbed  the  muscarin 
salt.  Nothnagel  separated  the  two  bases  as  platinochlorids  by  re- 
peated recrystallization  from  hot  water  and  washing  the  crystals 
with  cold  water.  The  platinochlorid  of  cholin  is  easily  soluble  in 
water. 

Physiological  Action.  —  Small  doses  of  this  ptomain  induce  in 
frogs  total  paralysis,  with  stoppage  of  the  heart  in  diastole,  and  this 


310  CHEMISTRY  OF  THE  PTOMAINS. 

action  is  antagonized  by  subsequent  injection  of  atropin.  In  the 
case  of  previously  atropinized  frogs  it  fails  to  antagonize.  Very 
small  doses  produced  in  rabbits  profuse  salivation  and  lachrymation, 
contraction  of  the  pupil,  profuse  diarrhea,  and  passage  of  urine  and 
semen ;  finally,  the  animal  died  in  convulsions,  which,  however,  were 
only  of  short  duration  (Brieger).  Although  the  natural  and  artificial 
muscarin  and  their  salts  are  chemically  and  physically  alike,  they 
are  not  however  identical,  although  so  considered  by  Schmiedeberg 
and  Harnack.  This  is  seen  in  their  physiological  action.  Thus 
Bohm  found  that  the  artificial  muscarin  paralyzed  intramuscular 
nerve  endings.  According  to  Meyer,  J^  -  J^  mg.  will  do  this, 
whereas  the  natural  base  will  not  have  this  effect.  Again,  1—2 
drops  of  a  1  per  cent,  solution  of  the  artificial  base  will  produce 
maximal  myosis  in  birds  in  a  few  minutes ;  while  the  natural  base 
has  no  effect  on  birds'  pupils.  The  action  of  betain  aldehyde  and 
iso-muscarin  has  already  been  stated.  Brieger' s  ptomain  would  seem 
to  be  nearly  identical  with  artificial  muscarin. 

Constitution  of  the  Members  of  the  Cholin  Group. — The  struc- 
ture of  cholin  was  clearly  demonstrated  by  Wurtz,  who  accomplished 
the  synthesis  of  this  base  by  treatment  of  ethylene  chlorhydrin  with 
trimethylamin.  This  same  method  can  be  applied  to  the  synthesis 
of  betain  and  neurin  by  using  monochloracetic  acid  and  vinylbromid 
instead  of  ethylene  chlorhydrin.  The  structural  formulae  which  can 
be  deduced  from  these  reactions  are  as  follows  : 


CHjOH 

gH, 

CO,H 

CH 

CH 

<fH, 

N(CH3)3.0H 
Cholin. 

l!r(CH3)3.0H 
Neurin. 

N(CH3)3.0H 
Betain. 

All  these  bases,  since  they  can  be  prepared  from  cholin,  may  also 
be  considered  as  oxidation  products  of  trimethyl-ethyl-ammonium 
hydrate : 

CH, 

N(CH3)3.0H. 


The  constitution  of  muscarin  is  still  unsettled.  Schmiedeberg 
and  Harnack  believed  that  it  resulted  from  cholin  by  the  oxidation 
of  hydrogen  connected  with  the  same  carbon  as  the  hydroxyl  group. 
Its  formula  would  be  either  (1)  or  (2),  as  given  below.  The  former, 
analogous  to  chloral  would  have  the  somewhat  rare  condition  of  two 
hydroxyl  groups  attached  to  one  carbon  atom.  The  presence  of 
hydroxyl  groups  in  muscarin  cannot,  however,  be  demonstrated  with 
acetic  anhydrid,  or  benzoyl  chlorid  (Nothnagel).  The  second  formula 
is  that  of  betain  aldehyd. 


MYDATOXIN.  311 

The  formulae  of  several  muscarin  compounds  are  herewith  presented  : 

(1)  (2)  (3) 

CH(OH),  COH  CPIjOH 


JH.^  CH,  CHOH 

N(CH,)3.0H        N(CH3)3.0H        N(CH3)3.0H. 
Muscarin.  Betain  Aldehyd.         Iso-muscaein. 

It  will  be  observed  that  very  slight  diflPerences  in  the  chemical 
constitution  of  cholin,  muscarin,  betain  and  neurin  are  accompanied 
by  very  great  differences  in  the  physiological  action  of  these  bases. 
Thus,  as  pointed  out  by  Schmidt,  cholin  may  be  considered  as  a  pri- 
mary alcohol  and  betain  as  a  monobasic  acid.  Between  these  two 
relatively  non-poisonous  bases  is  the  intermediate  oxidation  product, 
the  aldehyde  muscarin,  which  is  highly  poisonous. 

Again,  it  will  be  remembered  that  the  artificial  muscarin,  formed  by 
the  oxidation  of  cholin,  had  a  markedly  different  physiological  effect 
on  the  intramuscular  nerve  endings  and  on  the  pupils  of  birds  than 
the  natural  muscarin.  This  difference  must  undoubtedly  be  ascribed 
to  difference  in  stereochemical  structure,  as  in  the  case  of  active  and 
inactive  lactic  acids,  and  of  atropin.  Furthermore,  Bode's  iso- 
muscarin  possesses  likewise  an  entirely  different  action,  differing  only 
in  the  position  of  the  hydroxyl  group.  The  same  is  true  of  betain 
aldehyd,  which  differs  from  muscarin  by  the  elements  of  water. 

Mydatoxin,  CgH^jNOj. — This  base  was  obtained  by  Brieger  in 
1886  (III.,  25,  32)  from  several  hundred  pounds  of  human  internal 
organs  which  were  allowed  to  stand  in  closed  but  spacious  wooden 
barrels  for  four  months,  at  a  temperature  varying  from  —  9°  to  -|-  5°. 
He  obtained  much  larger  quantities  of  it,  however,  from  horse-flesh 
which  had  putrefied  under  the  same  conditions.  In  the  process  of 
extraction  it  is  found  in  the  mercuric  chlorid  precipitate  together 
with  cadaverin,  putrescin  and  another  base,  C^Hj^NOg.  It  can  be 
isolated  from  this  mixture  by  recrystallizing  the  mercury  salts,  which 
removes  the  cadaverin  because  of  its  difficult  solubility  in  water, 
and  decomposing  the  soluble  mercury  salts  by  hydrogen  sulphid. 
The  filtrate  freed  from  mercury  is  now  evaporated  to  dryness  and 
the  residue  repeatedly  extracted  with  absolute  alcohol,  in  order  to 
remove  the  putrescin  hydrochlorid  which  is  insoluble.  The  alco- 
holic solution,  after  standing  some  time  to  permit  complete  separation 
of  any  dissolved  putrescin,  is  then  evaporated  to  dryness  and  taken 
up  with  water.  This  solution  gives,  on  the  addition  of  gold  chlorid, 
a  precipitate  of  the  aurochlorid  of  the  base  C^IIj^N02.  The  filtrate 
from  this  precipitate,  containing  the  mydatoxin,  is  treated  with 
hydrogen  sulphid  to  remove  the  gold,  and  then  evaporated  to  dry- 
ness. The  colorless,  syrupy  hydrochlorid  thus  obtained  forms  with 
platinum  chlorid  a  double  salt  which  is  readily  soluble  in  water,  and 


312  CHEMISTRY  OF  THE  PTOMAINS. 

can  be  purified  by  repeated  recrystallization  from  absolute  alcohol 
containing  some  hydrochloric  acid. 

The  name  mydatoxin  is  derived  from  fioddw,  to  putrefy.  The 
free  base  is  obtained  from  the  hydrochlorid,  by  treatment  with  moist 
freshly  precipitated  silver  oxid,  as  a  strongly  alkaline  syrup  which 
solidifies  in  vacuo  to  plates.  It  is  insoluble  in  alcohol,  ether,  etc.  It 
does  not  distil  without  decomposition.  It  is  isomeric  with  leucin  and 
also  with  the  base  CgH^jNOg ,  obtained  by  Brieger  in  1 888  from  tetanus 
cultures.  It  is  isomeric  with  di-methyl  ethyl  betain  and  also  with 
tri-methyl  propiony]  betain  (Wildstatter,  Ber.,  35,  606,  610).  The 
latter  is  represented  by  formula  (1).    Both,  however  form  gold  salts. 

The  hydrochlorid,  CgH^gNOg.HCl,  is  a  colorless  deliquescent  syrup 
which  does  not  form  any  double  salt  with  gold  chlorid.  With  plat- 
inum chlorid  it  gives  an  easily  soluble  salt.  Otherwise  it  combines 
only  with  phosphomolybdic  acid,  with  which  it  forms  cubes.  Fer- 
ric chlorid  and  potassium  ferricyanid  yield,  after  a  time,  Berlin  blue. 
It  is  readily  soluble  in  alcohol. 

The  platinochlorid,  (C6Hj3N02.HCl)2PtCl,  (Pt  =  29.00  per  cent.), 
melts  at  193°,  with  decomposition.  It  crystallizes  in  plates  which 
are  extremely  soluble  in  water.  It  can  be  readily  recrystallized 
from  absolute  alcohol  acidulated  with  hydrochloric  acid.  The  mer- 
cury salt  is  readily  soluble  in  water. 

The  exact  formula  of  this  base,  of  mytilotoxin,  and  some  other 
bases,  cannot  be  considered  to  be  permanently  settled,  inasmuch  as 
the  formula  of  the  hydrochlorid,  CgH^gNOg.HCl,  as  deduced  from 
the  analysis  of  the  platinum  double  salt,  may  equally  apply  to  the 
base  CgHj^NOg.OH  as  to  the  base  CgH^gNOg.  If  the  first  formula 
is  correct,  then  mydatoxin  may  be  considered  a  homologue  of  betain, 
and  its  structure  would  be  expressed  by  (1).  Or,  it  may  have  the 
structure  shown  in  formula  (2). 

(2) 

CO.  OH 
H.CHj 

Qg  ]S^(CH3)3.0H. 

By  loss  of  water  in  either  case  (propyl  or  iso-propyl  betain)  the 
formula  CgHj3N02  results.  In  the  latter  instance  the  anhydrid  would 
be  that  which,  combined  with  pyridin,  is  met  with  in  pilocarpin. 

This  ptomaiu,  although  it  possesses  toxic  properties,  is  not,  however, 
a  strong  poison.  Its  action  is  the  same  as  that  of  the  base  C^H^^NOg 
(see  page  318),  with  which  it  is  associated,  except  that  the  symptoms 
of  poisoning  develop  slower,  so  that  the  death  of  a  guinea-pig  does 
not  take  place  for  about  twelve  hours.  White  mice  are  very  suscep- 
tible to  the  action  of  these  two  poisons.     A  short  time  after  the 


MYTILOTOXIK  313 

injection  of  even  small  doses  they  are  taken  with  convulsions  which 
come  on  in  paroxysms.  The  eyeballs  roll  upward.  Lachrymation, 
diarrhoea,  and  dyspnoea  develop  and  the  mice  die  within  a  short  time. 

A  Base  (?),  CgHjgNOg ,  an  isomer  of  the  preceding  and  also  of  leucin, 
was  obtained  by  Brieger  in  1888  from  tetanus  cultures.  It  is  not 
poisonous — distinction  from  mydatoxin.  It  probably  is  an  amido  acid. 
The  platinochlorid  crystallizes  in  plates,  is  easily  soluble  in  water  and 
in  alcohol,  and  melts  at  197°  with  decomposition  (see  page  322). 

Mytilotoxm,  CgIIj.N02 ,  is  the  specific  poison  of  toxic  mussel  (My- 
tilus  edulis),  from  Avhich  it  was  obtained  by  Brieger  in  1885  (III.,  76). 
This  poison  is  formed  during  the  life  of  the  animal  under  certain  condi- 
tions which  have  been  thoroughly  studied  by  Schmidtmann,  Virchow, 
and  others  (see  p.  192).  Brieger  obtained  the  poison  by  extracting 
the  toxic  mussel  with  acidulated  water,  and  evaporating  this  solution 
to  a  syrupy  consistency.  The  residue  was  thoroughly  extracted  with 
alcohol,  and  this  solution  was  treated  with  lead  acetate  in  order  to 
remove  mucilaginous  substances.  The  filtrate  was  then  evaporated 
and  the  residue  extracted  with  alcohol.  Any  lead  that  had  dissolved 
was  removed  by  hydrogen  sulphid.  The  alcohol  was  expelled,  and 
the  resulting  syrup  was  taken  up  with  water  and  decolored  by  boil- 
ing with  animal  charcoal.  The  clear  solution  was  now  neutralized 
with  sodium  carbonate,  acidulated  with  nitric  acid,  and  precipitated 
with  phosphomolybdic  acid.  The  precipitate  was  decomposed  by 
warming  with  neutral  lead  acetate,  and  the  resulting  filtrate,  after 
the  removal  of  the  lead  by  hydrogen  sulphid,  was  acidulated  with 
hydrochloric  acid  and  evaporated  to  dryness.  The  residue  was 
extracted  with  absolute  alcohol,  whereby  betain,  on  account  of  its 
insolubility,  is  removed,  and  the  alcoholic  solution  was  precipitated 
by  alcoholic  mercuric  chlorid.  The  mercury  precipitate  was  repeat- 
edly recrystallized  from  water  and  the  poison  thus  obtained  as  an 
easily  soluble  double  salt. 

The  free  base  as  obtained  by  the  addition  of  alkali  to  the  hydro- 
chlorid  possesses  a  disagreeable  odor  which  disappears  on  exposure 
to  air,  and  the  substance  ceases  to  possess  poisonous  properties. 
Brieger  has  proposed  the  application  of  this  test  for  the  recognition 
of  poisonous  mussel ;  on  treatment  of  these  with  alkali  the  charac- 
teristic odor  is  developed.  Mytilotoxin  is  also  destroyed  on  distilla- 
tion with  potassium  hydrate,  and  in  the  distillate  there  is  found  an 
aromatic  non-poisonous  product  and  trimethylamin.  The  free  base, 
therefore,  does  not  exist  by  itself  for  any  length  of  time  but  soon 
becomes  converted  into  an  inert  substance.  H.  Salkowski  has  also 
shown  that  it  is  destroyed  on  boiling  with  potassium  carbonate, 
whereas  its  hydrochloric  acid  solution  can  be  evaporated  to  dryness 
and  heated  to  110°  without  destroying  its  poisonous  property. 


314  CHEMISTRY  OF  THE  PTOMAINS. 

The  hydrochlorid,  CgHjgNOg.HCl,  prepared  from  the  aurochlorid, 
crystallizes  in  tetrahedra.  It  is  extremely  poisonous,  and  according 
to  Brieger  produces  exactly  the  same  symptoms  which  have  been  ob- 
served by  Schmidtmann  in  persons  who  have  partaken  of  poisonous 
mussels  (see  page  190).  On  standing,  however,  the  pure  hydrochlorid 
gradually  becomes  dark  and  decomposes  with  loss  of  its  poisonous 
property — a  change  corresponding  to  that  which  tetanin  undergoes 
(p.  322).  The  gold  salt  is  better  adapted  for  preservation.  The 
ordinary  alkaloidal  reagents  produce  in  its  solutions,  if  at  all,  only 
oily  precipitates. 

The  aurochlorid,  CgHi^NO^.HCl.AuClj  (Au  =  41.66  per  cent.), 
crystallizes  in  cubes.     Its  melting  point  is  182°. 

It  is  well  to  observe  that  Brieger  was  unable  to  obtain  this  base 
from  mussels  that  were  allowed  to  putrefy  for  sixteen  days. 

As  stated  under  mydatoxin,  the  formula  of  the  hydrochlorid, 
CgHj.NO2.HCl,  is  applicable  to  either  one  of  the  two  bases,  CgH^g 
NO2.OH  or  CpHjgNOg .  The  base  corresponding  to  the  first  formula 
is  evidently  a  homologue  of  muscarin,  and  should  possess  a  similar 
physiological  action.  As  a  matter  of  fact,  mytilotoxin  does  resemble 
muscarin  somewhat  in  this  respect,  and  its  occurrence  together  with 
betain  would  seem  to  make  it  a  decomposition  product  of  lecithin,  in 
which  case  this  base  must  be  looked  upon  as  a  member  of  the  cholin 
group.  A  compound  corresponding  to  the  formula  CgHjgNOgOH 
was  prepared  by  Hanriot  in  a  manner  analogous  to  Wurtz's  synthesis 
of  cholin,  by  treating  glycerin  monochlorhydrin  with  trimethylamin. 
This  base,  trimethyl-glyceryl  ammonium  hydrate,  has  the  structure  : 


1 


H^.CHOHCHjOH 

N(CH3)30H. 


It  was  suggested  that  Hanriot's  base  might  possibly  be  identical 
with  mytilotoxin,  but  a  careful  comparison  made  by  Brieger  showed 
that  it  possesses  no  physiological  action  and  that  its  chemical  prop- 
erties are  entirely  difierent. 

Mytilotoxin  would,  therefore,  seem  to  possess  the  formula  CgHj^ 
NOg,  as  originally  given  by  Brieger.  From  the  fact  that  on  distilla- 
tion with  potassium  hydrate  it  yields  trimethylamin,  it  follows  that 
mytilotoxin  is  a  quaternary  base.  He  is  inclined  to  regard  it  as  a 
methyl  derivative  of  betain,  which  is  so  common  in  mussels,  and 
represents  it  by  formula  No.  1. 

(1)  (2) 

CO2H  CO.H 

CH.CH,  CH.CHj 

N(CH3)3.0H  N(CHs),.OH 


GADININ.  315 

No.  1,  however,  is  CgH^gNOj,  instead  of  CgHj^NOg,  as  above, 
and  has  been  referred  to  under  mydatoxin.  The  formula  No.  2, 
CgH^jNO^,  may  be  taken  to  represent  more  correctly  this  base.  On 
comparing  these  formulae  it  would  appear  that  mydatoxin  (1)  is  an 
oxidation  product  of  mytilotoxin  (2).  The  latter  in  turn  would  be  a 
homologue  of  betain  aldehyde  (p.  311),  and  hence  a  derivative  of 
cholin  or  muscarin. 

According  to  Brieger,  mytilotoxin  produces  all  the  characteristic 
effects  seen  in  mussel  poisoning.  In  its  paralyzing  action  it  resem- 
bles curara.  This  action  is  explainable  now  that  Glause  and  Luch- 
singer  have  shown  that  all  trimethyl  ammonium  bases  have  a  mus- 
carin-like  action.  For  the  symptoms  induced  by  poisonous  mussel 
see  page  190. 

Gadinin,  C^Hj^NOj,  was  found  in  haddock  (1885)  which  were 
allowed  to  decompose  in  open  iron  vessels  for  j&ve  days  during  sum- 
mer. Brieger  also  obtained  it  from  cultures  of  the  bacteria  of 
human  feces  on  gelatin.  Carbone  found  it  in  cultures  of  the 
Proteus  vulgaris.  The  decomposing  mass  was  thoroughly  stirred 
every  day  in  order  to  bring  it  into  contact  with  atmospheric  oxygen 
(Brieger,  I.,  49).  It  was  then  treated  with  water,  and  hydrochloric 
acid  was  added  to  acid  reaction.  After  being  warmed  the  mixture 
was  filtered  and  the  filtrate  concentrated  on  the  water-bath  to  a 
syrupy  consistency.  This  syrupy  residue  was  extracted  with  water, 
and  the  aqueous  solution  was  precipitated  with  mercuric  chlorid. 
This  mercuric  chlorid  precipitate  contained  a  base,  the  quantity 
of  which,  however,  was  insufficient  for  a  complete  analysis  (see 
page  325).  The  mercuric  chlorid  filtrate,  after  the  removal  of 
the  mercury  by  hydrogen  sulphid,  was  evaporated  to  a  syrup, 
and  this  was  then  repeatedly  extracted  with  alcohol.  The  alco- 
holic solution  thus  obtained  contained  neuridin,  a  base  of  the  same 
composition  as  ethylendiamin,  muscarin,  gadinin,  and  triethyl- 
amin.  These  bases  were  separated  in  the  following  manner  :  The 
alcoholic  solution  gave  with  platinum  chlorid  a  precipitate  of  neuridin. 
The  filtrate  from  this  platinum  precipitate  was  heated  on  the  water- 
bath  to  expel  the  alcohol  after  which  the  platinum  was  removed  by 
hydrogen  sulphid.  The  aqueous  filtrate  was  concentrated  to  a  small 
volume  which,  on  addition  of  platinum  chlorid,  gave  a  precipitate 
of  the  isomer  of  ethylendiamin.  The  mother-liquor  from  this  pre- 
cipitate was  concentrated  on  a  water-batb,  and  on  cooling  the  platino- 
chlorid  of  muscarin  crystallized  out.  From  the  mother-liquor  of 
this  precipitate  on  standing  in  a  desiccator,  the  gadinin  double  salt 
crystallized.  The  mother-liquor  from  the  gadinin  platinochlorid  was 
treated  with  hydrogen  sulphid  to  remove  the  platinum,  and  the 
aqueous  filtrate  on  distillation  with  potassium  hydrate  gave  triethyl- 
amin. 


316  CHEMISTRY  OF  THE  PTOMAINS. 

Gadinin  (from  Gadus  callarias,  haddock)  in  small  doses  does  not 
appear  to  be  poisonous  ;  large  doses  (0.5-1  gram)  are  decidedly  toxic 
and  may  kill  guinea-pigs.  The  formula  of  the  free  base  as  deduced 
from  the  analysis  of  the  platinochlorid  may  be  either  C-Hj^NOg  or 
C\H,3NO,.OH. 

The  hydrochlorid,  C^Hj^NOg.HCl,  as  obtained  by  the  decomposi- 
tion of  the  platinochlorid  with  hydrogen  sulphid,  crystallizes  under 
the  desiccator  in  thick,  colorless  needles,  which  are  easily  soluble  in 
water  ;  insoluble  in  alcohol.  It  forms  no  combination  with  gold 
chlorid,  but  does  give  crystalline  precipitates  with  phosphomolybdic, 
phosphotungstic  and  picric  acids. 

The  platinochlorid,  (C7Hi7N02.HCl)2PtCl,(Pt  =  27.68  per  cent.), 
is  at  first  quite  soluble,  and  on  standing  in  a  desiccator  it  crystallizes 
in  golden-yellow  plates  which,  when  once  formed,  are  again  difficultly 
soluble  in  water.  It  can  be  recrystallized  from  hot  water.  It  melts 
at  214°. 

Typhotoxin,  C^Hj^]S'02. — This  base  was  named  thus  by  Brieger 
in  1885  (III.,  86),  and  was  regarded  by  him  as  the  specific  toxic 
product  of  the  activity  of  Koch-Eberth's  typhoid  bacillus.  It  is 
however,  certain,  as  in  the  case  of  tetanus,  that  the  real  poison  of  this 
germ  is  not  this  ptomain,  but  rather  a  toxin.  He  obtained  the  base  by 
cultivating  the  bacillus  on  beef-hash  for  eight  to  fourteen  days  at 
37.5°- 38°.  The  nature  of  the  soil  on  which  the  bacillus  grows  has 
a  great  deal  to  do  with  the  formation  of  the  poison.  An  espe- 
cially important  factor  is  the  temperature,  for  Brieger  observed  that 
no  poison  was  produced  in  one  case  where  the  temperature  remained 
by  accident  at  39°  for  twenty- four  hours.  Under  such  conditions 
creatin  is  present  in  quantity,  whereas  otherwise  the  reverse  is  the  rule. 

In  the  process  of  extraction  (p.  232)  it  occurs  in  the  mercuric  chlo- 
rid precipitate,  and  from  this  it  is  obtained,  after  the  removal  of  the 
mercury  by  hydrogen  sulphid,  as  an  easily  deliquescent  hydrochlorid. 
This  for  the  purpose  of  purification  is  converted  into  the  difficultly 
soluble  aurochlorid. 

Typhotoxin  is  isomeric  with  gadinin  and  the  compound  CyHj^NOg 
which  Brieger  obtained  from  putrefying  horse-flesh.  In  its  properties 
it  is,  however,  very  different.  Thus,  the  free  base  is  strongly  alka- 
line, and  its  hydrochlorid  yields  a  difficultly  soluble  picrate.  On  the 
other  hand,  the  isomer  from  horse-flesh  possesses  a  slightly  acid 
reaction,  and  does  not  form  a  picrate.  Again,  typhotoxin  gives  with 
Ehrlich's  reagent  (sulphodiazobenzol)  an  immediate  yellow  color, 
which  disappears  upon  the  addition  of  alkali,  whereas  the  isomer  does 
not  yield  this  reaction.  Furthermore,  the  two  bases  differ  in  their 
physiological  action  and  in  their  behavior  to  alkaloidal  reagents  (see 
Table  I.).  Their  aurochlorids,  however,  possess  the  same  melting- 
point. 


A  BASE.  317 

The  hydrochlorid  is  readily  deliquescent,  and  unites  with  platinum 
chlorid  to  form  an  easily  soluble  double  salt  crystallizing  in  needles. 

The  aurochlorid,  C^H^.NO^.HCl.AuClj  (Au  =  40.46  per  cent.),  is 
difficultly  soluble,  and  crystallizes  in  prisms,  which  melt  at  176°.  In 
its  melting-point  and  solubility  (197°,  Brieger,  Arch.  f. pathol.  Anat., 
115,  489)  it  agrees  with  its  isomer  from  horse-flesh.  In  his  first  ex- 
periments with  the  typhoid  bacillus,  Brieger  (II.,  69)  obtained  a  basic 
product  diifering  in  some  of  its  characters  from  typhotoxin.  Its 
aurochlorid,  on  analysis,  gave  41.91  and  41.97  per  cent,  of  Au ;  16.06 
per  cent,  of  C;  and  3.66  per  cent,  of  H;  while  typhotoxin  aurochlorid 
gave  40.78  per  cent.  Au;  17.38  per  cent.  C;  and  3.85  per  cent.  H. 
For  a  comparison  of  the  reaction  of  these  two  substances,  see  Table  I. 

In  its  physiological  action  typhotoxin  differs  from  its  isomer 
(page  318)  in  that  the  latter  produces  symptoms  with  well  marked 
convulsions,  whilst  the  former  throws  the  animal  into  more  of  a 
paralytic  or  lethargic  condition.  The  action  of  this  base  has  been 
studied  only  on  mice  and  guinea-pigs.  It  produces  at  first  slight 
salivation  with  increased  respiration  ;  the  animals  lose  control  over 
the  muscles  of  the  trunk  and  extremities  and  fall  down  helpless 
upon  their  sides.  The  pupils  become  strongly  dilated  and  cease  to 
react  to  light;  the  salivation  becomes  more  profuse  ;  the  rate  of  heart- 
beat and  of  respiration  gradually  decreases  and  death  follows  in  from 
one  to  two  days.  Throughout  the  course  of  these  symptoms  the  ani- 
mals have  frequent  diarrhoeic  evacuations,  but  at  no  time  are  con- 
vulsions present.  On  post  mortem  the  heart  is  found  to  be  in 
systole,  the  lungs  are  strongly  hypersemic,  the  other  internal  organs 
pale,  the  intestines  firmly  contracted  and  their  walls  pale. 

A  Base  (?),  C^Hj^NO^,  was  obtained  by  Brieger  in  1886  (III.,  28) 
on  working  over  about  one  hundred  pounds  of  horse-flesh  which  had 
been  allowed  to  undergo  slow  putrefaction  with  limited  access  of  air 
and  at  a  low  temperature  ( —  9°  to  +  5°)  for  four  months.  It  occurred 
in  the  mercuric  chlorid  precipitate  together  with  cadaverin,  putrescin 
and  mydatoxin,  and  from  these  bases  it  was  separated  and  isolated 
according  to  the  method  given  on  page  311. 

A  similar,  if  not  identical,  substance  having  the  composition 
CylljyNO,,  was  obtained  by  Baginsky  and  Stadthagen  (1890)  from 
cultures  on  horse-flesh,  ten  days  at  35°,  of  a  bacillus  closely  allied 
to  Finkler-Prior's  and  isolated  from  stools  of  cholera  infantum. 
The  gold  salt  in  its  crystalline  form  and  properties  was  the  same  as 
Brieger's  except  that  it  possessed  a  somewhat  higher  melting-point. 
It  is  possible  that  one  or  more  of  these  isomers  are  homologues  of  leucin. 

The  free  substance  possesses,  even  after  most  careful  purification, 
a  slightly  acid  reaction.  This  acidity  is  removed  from  even  a  large 
quantity  of  the  substance  by  the  addition  of  a  drop  of  alkali.  On 
account  of  the  acid  character  of  the  free  substance  Brieger  does  not 


318  CHEMISTRY  OF  THE  PTOMAINS. 

consider  it  to  be  a  base  (a  ptomain).  It  differs,  however,  from  the 
amido  acids  in  its  poisonous  character ;  in  the  fact  that,  unlike  an 
acid,  it  does  not  unite  with  bases  to  form  salts ;  and  in  not  giving 
the  characteristic  red  coloration  (Hofmeister's  reaction  for  the  amido 
acids)  with  ferric  chlorid.  Whatever  the  true  nature  of  this  sub- 
stance may  be,  it  nevertheless  in  its  other  properties  behaves  like  a 
base.  Thus,  it  forms  simple  as  well  as  double  salts.  On  boiling 
with  copper  acetate  it  gives  amorphous  floccules.  Under  the  desic- 
cator it  solidifies  into  plates  which  deliquesce  on  exposure  to  the 
air.  It  does  not  combine  either  with  silver  oxid  or  with  cupric 
hydrate.  On  dry  distillation  it  yields  a  distillate  possessing  a  strong 
acid  reaction  and  a  peculiar  odor.  The  distillate  does  not  give  any 
precipitate  with  platinum  chlorid,  or  with  gold  chlorid,  nor  does  it 
react  with  copper  acetate.  With  phosphomolybdic  acid,  however,  it 
forms  an  amorphous  mass ;  with  ferric  chlorid  and  potassium  ferri- 
cyanid  it  yields  an  immediate  precipitate  of  Berlin  blue,  whereas  the 
original  substance  does  not  give  any  blue  coloration. 

The  hydrochlorid,  C^H^yNOj.HCl,  crystallizes  in  fine  needles  which 
are  insoluble  in  absolute  alcohol.  When  the  aqueous  solution  is 
treated  with  freshly  precipitated  silver  oxid  the  resulting  filtrate  con- 
tains in  solution  some  silver  oxid,  which  can  be  removed  by  hydrogen 
sulphid;  thus  differing  from  an  ammoniacal  silver  solution,  which 
gives  no  precipitate  on  treatment  with  hydrogen  sulphid.  In  this 
respect  it  resembles  Salkowski's  base,  page  287.  For  reactions  of 
the  hydrochlorid,  see  Table  I. 

The  aurochlorid,  Cyllj^NOg.HCl.AuClg,  forms  plates  which  are 
difficultly  soluble  in  water  and  melt  at  176° — the  melting-point  of 
the  gold  salt  of  typhotoxin.  It  is  dimorphous,  since  sometimes  it  is 
also  obtained  in  needles  which  can  be  changed  into  plates. 

It  does  not  form  a  picrate,  nor  does  it  give  a  reaction  with  sulpho- 
diazobenzol. 

This  substance,  when  injected  into  frogs,  produces  a  curara-like 
action.  A  few  minutes  after  the  injection  the  animal  falls  into  a 
condition  of  paralysis  and,  although  it  can  still  react  toward  reflexes, 
it  cannot  move  from  its  place.  At  times  fibrillary  twitchings  pass 
over  the  body.  The  pupils  dilate,  the  heart-action  becomes  gradually 
weaker,  and  finally,  after  several  hours,  the  animal  dies  with  the 
heart  in  diastole.  Doses  of  0.05  to  0.3  gram  of  the  hydrochlorid, 
injected  into  guinea-pigs,  produce  in  a  short  time  a  slight  tremor, 
gradual  increase  in  respiration,  and  slight  moistening  of  the  lower 
lip.  The  pupils  at  first  contract,  then  dilate  ad  maximum  and  be- 
come reactionless.  The  temperature  remains  at  first  normal ;  chills 
of  short  duration  follow  in  rapid  succession.  The  animal  squats  on 
the  ground,  with  its  snout  pressing  against  the  floor  in  exactly  the 
same  way  as  in  the  case  of  mussel-poison.  Violent  clonic  convul- 
sions follow  in  continually  shorter  intervals,  and  at  the  same  time 


MORRHUIG  ACID.  319 

lachrymation  and  salivation  become  profuse,  but  not  so  excessive  as 
in  the  case  of  the  muscarin-like  ptomains.  The  temperature  sinks 
with  the  decrease  in  the  rate  of  respiration,  the  ears  previously- 
gorged  become  pale  and  cold,  and  the  heart-action  becomes  irregular 
and  less  frequent  than  before.  General  paralysis  sets  in,  but  the 
head  still  moves  upward  and  backward.  External  stimuli  induce 
violent  clonic  convulsions,  the  animal  repeats  frequently  choking 
movements,  and  at  the  same  time  yields  large  quantities  of  saliva ; 
finally,  it  falls  upon  its  side  completely  paralyzed  and  dies.  The 
heart  stops  in  diastole,  the  intestines  are  pale  and  strongly  contracted, 
and  the  bladder  is  empty  and  contracted. 

Morrhuic  Acid,  CgHjgNOg ,  was  obtained  by  Gautier  and  Mour- 
gues  (1888)  from  brown  cod-liver  oil,  together  with  six  bases  already 
described — namely,  butylamin,  araylamin,hexylamin,  dihydrolutidin, 
asellin,  and  morrhuin.  These  bases  constitute  about  0.2  per  cent,  of 
the  oil.  The  discoverers  regard  them  as  true  leucomains,  dissolved 
from  the  hepatic  cells  by  the  oil.  Bouillot  found  that  the  mixed 
bases,  or  total  basic  product,  in  a  dose  of  0.25-0.15  g.  in  man  increased 
the  volume  of  urine  and  the  quantity  of  urea.  By  a  microchemical 
reaction,  exposing  sections  of  liver  to  the  fumes  of  hydrochloric  or 
hydrofluoric  acid,  he  detects  the  bases  in  the  liver,  especially  in  the 
bile-ducts.  The  bases,  therefore,  exist  preformed  in  the  cod's  liver, 
and  are  derived  from  the  bile.  It  is  more  probable,  however,  that 
these  compounds  are  the  products  of  initial  decomposition,  and  for 
that  reason  they  are  described  under  the  head  of  ptomains. 

This  compound  is  relatively  abundant,  and  is  basic  as  well  as  acid 
in  character.  It  is  resinous  in  appearance,  and  can  be  crystallized 
in  flattened  prisms,  or  large  lance-shaped  plates.  When  freshly 
precipitated  it  is  oleaginous,  viscous,  then  gradually  hardens.  It 
possesses  a  disagreeable  aromatic  odor  resembling  that  of  the  sea- 
weeds upon  which  the  fish  feed.  According  to  the  discoverers,  its 
probable  source  is  the  lecithin  derived  thus  from  these  weeds.  It  is 
soluble  in  alcohol,  and  but  slightly  in  ether.  It  reddens  turmeric, 
decomposes  carbonates,  and  with  acids  forms  salts  which  precipitate 
lead  acetate  and  silver  nitrate,  but  not  copper  acetate,  even  on  warming. 

The  hydrochlorid  is  crystalline  and  is  partially  dissociated  by 
excess  of  water.  The  platinum  salt  is  soluble  and  crystallizes  in 
very  small  cross-shaped  prismatic  needles.  The  gold  salt  is  amor- 
phous and  is  readily  altered  on  heating. 

The  properties  of  this  compound  show  that  it  is  of  a  pyridin 
nature,  and  inasmuch  as  it  does  not  give  a  precipitate  with  copper 
acetate,  it  would  appear  that  the  carboxyl  is  not  directly  united  to 
the  pyridin  nucleus.  This  does  not  necessarily  follow,  now  that  we 
know  that  some  amido  acids  exist  which  do  not  give  a  reaction  with 
copper  acetate  (see  page  287).     Its  pyridin  nature  is  furthermore 


320  CHEMISTRY  OF  THE  PTOMAINS. 

shown  on  distillation  with  lime.  An  oily  alkaline  base  is  thus 
obtained  which  forms  an  iodomethylate,  and  this  with  potassium 
hydrate  yields  quite  an  intense  red  color,  resembling  lees  (de 
Coninck's  reaction).  On  oxidation  with  permanganate  of  potassium 
it  yields  a  monobasic  acid.  According  to  Gautier  and  Mourgues, 
the  compound  is  probably  identical  with  de  Jungh's  gaduin,  and  they 
ascribe  to  it  the  following  constitution  which,  it  should  be  said, 
lacks  confirmation. 

H 

/\ 

HC         COH 


H 


A  Base,  C^^^p^,  was  obtained  by  Pouchet  (1884)  from  the 
residual  liquors  resulting  from  an  industrial  treatment  of  debris  of 
bones,  flesh,  and  waste  of  all  kinds,  with  dilute  sulphuric  acid.  It 
is  accompanied  by  another  base,  CyH^glSTgOg ,  from  which  it  can  be 
separated  by  treatment  with  alcohol.  The  base  itself  forms  tufts 
of  delicate  needles  which  alter  or  decompose  less  easily  than  the 
accompanying  base.  The  platinochlorid,  (Q^^^f) ^J^C\)^iC\, 
forms  a  dull  yellow  powder  somewhat  soluble  in  strong  alcohol,  but 
insoluble  in  ether.  The  platinochlorid,  {C^'R^^f>^.llQ\)^iQ\,  is 
insoluble  in  ether. 

The  hydrochlorids  of  these  bases  form  silky  needles,  which  are 
altered  by  excess  of  hydrochloric  acid  and  by  exposure  to  air. 
Pouchet  considers  them  to  be  closely  allied  to  the  oxybetains.  The 
general  alkaloidal  reagents  precipitate  these  bases ;  the  phospho- 
molybdic  precipitate,  on  addition  of  ammonia,  gives  a  blue  tint. 
Both  bases  are  toxic  and  exert  a  paralyzing  action  upon  the  reflex 
movements. 

The  method  employed  by  Pouchet  for  their  isolation  was  to  pre- 
cipitate them  as  tannates.  The  precipitate  was  decomposed  by  lead 
hydrate  in  the  presence  of  strong  alcohol,  the  excess  of  lead  removed 
from  the  solution  by  hydrogen  sulphid,  and  the  clear  liquid  thus  ob- 
tained was  submitted  to  dialysis.  The  above  bases  occurred  in  the 
dialysate.  In  the  non-dialyzable  portion  volatile  bases  were  found 
probably  identical  with  those  described  by  Gautier  and  Etard. 

Tetanin,  C^J.\^^f>^,  was  obtained  in  1886  by  Brieger  (III.,  94) 
by  cultivating  impure  tetanus  microbes  of  Rosenbach,  in  an  atmos- 
phere of  hydrogen  on  beef-broth  for  eight  days  at  37°- 38°.  It  like- 
wise occurs  in  cultures  on  brain-broth.  Later  (April,  1888),  Brieger 
succeeded  in  obtaining  tetanin  from  the  amputated  arm  of  a  tetanus 


TETANIN.  321 

patient,  identical  in  its  physiological  and  chemical  reactions  with  that 
isolated  from  cultures  of  Rosenbach's  germs  on  beef-broth.  The 
presence  of  tetanin  during  life  in  tetanus  patients  has  thus  been 
demonstrated.  It  has  not  been  found  in  the  brain  and  nerve  tissue 
of  persons  dead  from  tetanus.  A  portion  of  the  jelly-like  mass  taken 
from  the  amputated  arm  was  found  to  contain  tetanus  bacilli  as  well 
as  staphylococci  and  streptococci,  and  when  planted  on  beef-broth 
tetanin  w^as  formed,  but  no  tetanotoxin  or  spasmotoxin. 

Kitasato  and  Weyl  (1890),  employing  pure  cultures  of  the  tetanus 
bacillus,  obtained  from  1:^  kilograms  of  beef  used  as  culture  medium 
1.7118  gram  of  tetanin  hydrochlorid  (0.137  per  cent.).  Tetano- 
toxin was  also  present. 

For  its  isolation  Brieger  employed  the  following  method  :  The 
cultures  were  slightly  acidulated  with  hydrochloric  acid,  heated  and 
filtered ;  the  filtrate  was  then  treated  with  lead  acetate  and  with  alco- 
holic mercuric  chlorid  in  the  manner  described  under  mytilotoxin 
(page  313).  Kitasato  and  Weyl  digest  the  cultures  with  0.25  per 
cent,  hydrochloric  acid  for  some  hours  at  60°,  then  render  slightly 
alkahne,  filter,  and  distil  in  vacuo  at  60°.  The  residue  in  the  retort 
is  worked  for  tetanin  by  Brieger's  method,  while  the  distillate  con- 
tains tetanotoxin,  ammonia,  indol,  hydrogen  sulphid,  phenol,  and 
butyric  acid.  The  filtrate  from  the  above  mercuric  chlorid  precipitate 
contains  the  greater  part  of  the  active  principle,  provided  the  precipi- 
tate has  been  thoroughly  washed.  After  the  removal  of  the  mercury 
by  hydrogen  sulphid  the  liquid  is  evaporated,  and  the  residue  is  re- 
peatedly extracted  with  absolute  alcohol,  in  which  the  tetanus  poison 
readily  dissolves  and  can  thus  be  separated  from  the  insoluble  am- 
monium chlorid.  The  alcoholic  solution  is  treated  with  alcoholic 
platinum  chlorid  which  precipitates  the  ammonium  and  creatinin 
platinochlorids,  whilst  the  platinochlorid  of  the  poison  remains  in 
solution.  The  filtrate  from  this  precipitate  gives,  on  the  addition  of 
ether,  a  flocculent  precipitate  possessing  exceedingly  deliquescent 
properties.  The  precipitate  is,  therefore,  rapidly  filtered  off  by  means 
of  a  pump,  and  dried  in  vacuo.  It  can  then  be  recrystallized  from 
hot  96  per  cent,  alcohol,  and  the  beautiful  clear-yellow  plates  thus 
obtained,  if  dried  again  in  vacuo,  become  rather  difficultly  soluble  in 
water,  from  which  it  can  then  be  recrystallized  and  obtained  in  a  per- 
fectly pure  condition.  If  boiled  with  boneblack,  it  decomposes 
yielding  a  non-poisonous  crystalline  compound. 

Phosphomolybdic  acid  cannot  be  used  in  the  separation  of  tetanin 
inasmuch  as  it  destroys  the  poison  (Brieger).  Bocklisch  has  also 
observed  that  this  reagent  destroys  the  poison  formed  in  the  putre- 
faction of  fish. 

Tetanin  obtained  by  treating  the  hydrochlorid  with  freshly  pre- 
cipitated moist  silver  oxid  forms  a  strongly  alkaline  yellow  syrup. 
With  alkaloidal  reagents  it  gives  the  same  reactions  as  the  hydro- 
21 


322  CHEMISTRY  OF  THE  PTOMAINS. 

chlorid,  except  that  it  does  not  give  a  blue  color  with  ferric  chlorid 
and  potassium  ferricyanid.  It  is  easily  decomposed  in  acid  but  is 
permanent  in  alkaline  solution. 

The  hydrochloride  Cj3H3gN20^.2HCl,  is  very  deliquescent,  and  is 
easily  soluble  in  absolute  alcohol.  Besides  with  platinum  it  com- 
bines only  with  phosphomolybdic  acid  to  form  an  easily  soluble 
crystalline  precipitate,  which  on  the  addition  of  ammonium  hydrate 
becomes  white.  If,  however,  the  hydrochlorid  is  impure,  phospho- 
molybdic acid  produces  a  precipitate  which  is  colored  an  intense  blue 
by  ammonia.  Potassium  bismuth  iodid  yields  a  precipitate  which  is 
at  first  amorphous,  but  soon  becomes  crystalline.  Ferric  chlorid  and 
potassium  ferricyanid  produce  a  slowly  developing  blue  color,  which 
probably  is  due  to  impurities. 

When  kept  for  some  months  the  highly  poisonous  hydrochlorid 
becomes  syrupy,  brownish,  and  wholly  inert.  Examined  at  this 
stage,  the  syrup  was  found,  by  means  of  platinum  chlorid,  to  contain 
a  substance  the  hydrochlorid  of  which  crystallized  in  plates.  This 
is  readily  soluble  in  water  and  alcohol  and  melts  at  197°  with  total 
decomposition,  the  same  as  tetanin.  It  combines  only  with  phos- 
phomolybdic acid  to  form  an  easily  soluble  compound.  The  plati- 
num salt  has  the  composition  CgIIj3N02.2IICl.PtCl^.  This  sub- 
stance is  non-poisonous,  and  probably  is  an  amido  acid.  It  is  different, 
however,  from  leucin  and  Nencki's  isomers  of  leucin,  although  pos- 
sessing the  same  composition.  It  is  also  isomeric  with  mydatoxin, 
CgHjjNOg,  but  this  is  highly  poisonous  to  mice,  while  the  former  is 
inert  (see  p.  312).  Tetanin  resembles  mytilotoxin  with  respect  to 
this  loss  of  toxicity  on  standing. 

The  platinochlorid,  Ci3H3,N20,.2HCl.PtCl,  (Pt=  28.33  per  cent.), 
is  easily  soluble  in  absolute  alcohol,  from  which  it  is  precipitated  on 
the  addition  of  ether.  From  ninety-six  per  cent,  alcohol  it  crystal- 
lizes in  clear  yellow  plates.  After  repeated  recrystallization  from 
alcohol  and  drying  in  vacuo  it  becomes  difficultly  soluble  in  water  so 
that  it  can  be  recrystallized  from  the  latter.     It  decomposes  at  197°. 

This  base  produces  the  characteristic,  though  by  no  means  all 
the  symptoms  of  tetanus,  since  we  know  of  at  least  three  other 
toxins  (pp.  61,  254)  which  occur  with  tetanin  in  cultures  of  the 
tetanus  microbe.  The  symptoms  induced  by  relatively  large  doses 
in  warm-blooded  animals,  as  mice,  guinea-pigs,  and  rabbits,  exhibit 
two  distinct  phases.  In  the  first,  the  animal  is  thrown  into  a 
lethargic,  paralytic  condition,  then  suddenly  becomes  uneasy,  and  the 
respiration  becomes  more  frequent.  This  is  followed  by  the  second 
phase,  in  which  tonic  and  clonic  convulsions,  especially  the  former, 
predominate  till  death  results.  0.5  gram  has  but  slight  action  on 
guinea-pigs.  Small  doses  do  not  seem  to  affect  guinea-pigs,  while 
frogs  appear  to  be  much  less  sensitive  than  mice.  The  characteristic 
convulsions  and  opisthotonus  seen  in  tetanus  in  man  are  also  pro- 


tybotoxico:n.  323 

duced  in  guinea-pigs  on  injection  of  large  doses  of  this  base.     Dogs 
and  horses  seem  to  be  but  slightly  sensitive  to  the  action  of  this  poison. 

A  Base,  C^^^^Nfi^,  was  isolated  by  Guareschi  in  1887  from 
putrid  fibrin.  It  occurs  in  the  chloroform  or  ether  extracts  along 
with  the  base  0^^,11  jgN,  and  is  probably  an  amido  acid  (see  page  259). 

A  Base,  C^HjgN20g,  was  isolated  by  Pouchet  in  1884.  It  is  said 
to  form  short,  thick  prisms  which  become  brown  when  exposed  to  light. 

The  platinochlorid,  (C7HjgN20g.IICl)2PtCl^ ,  crystallizes  in  pris- 
matic needles  which  are  insoluble  in  strong  alcohol.  For  further 
details  in  regard  to  this  base,  see  page  320. 

A  Base,  CjgH23N2^^,  was  obtained  by  Lepierre  (1894)  in  small 
quantity  from  poisonous  cheese  by  precipitating,  in  the  cold,  with 
acetate  of  copper.  It  is  crystalline,  bitter,  inodorous,  and  shows 
a  slight  acid  reaction  to  phenol-phthalein ;  is  but  slightly  soluble  in 
water,  soluble  in  alcohol.  It  is  dextro-rotatory,  is  precipitated  by 
phosphomolybdic  and  picric  acids,  iodin  in  potassium  iodid  ;  not  by 
tannin.  When  fed  to  guinea-pigs  it  produced  diarrhoea.  0.05  g. 
injected  intravenously  into  a  rabbit  had  no  effect.  The  hydrochlorid 
is  very  soluble  and  forms  large  needles.  The  platinum  and  gold 
salts  are  crystalline. 

Tyrotoxicon  has  been  found  in  poisonous  cheese  (Vaughan, 
Wallace,  Wolff,  Wesener  1898),  in  poisonous  ice  cream  (Vaughan, 
Novy,  Schearer,  Ladd),  in  poisonous  milk  (Vaughan,  Novy,  Newton, 
Wallace,  Firth,  Schearer),  and  in  cream  puffs  (Stanton).  The 
method  of  separating  this  poison  and  its  effect  upon  animals  have 
already  been  given  with  sufficient  detail.  Chemically,  it  is  very 
instable.  When  warmed  with  water  to  about  90°,  it  decomposes. 
Hydrogen  sulphid  also  decomposes  it,  therefore  all  attempts  to 
isolate  it  by  precipitation  with  some  base,  such  as  mercury  or  lead, 
and  then  removing  the  base  with  hydrogen  sulphid  have  failed.  Its 
unstable  character  is  illustrated  by  the  fact  that  it  may  disappear 
altogether  within  twenty-four  hours  from  milk,  rich  in  the  poison, 
which  is  allowed  to  stand  in  an  open  beaker. 

With  potassium  hydrate  it  forms  a  compound  which  agrees  in 
crystalline  form,  chemical  reactions,  and  the  per  cent,  of  potassium 
which  it  contains,  with  the  compound  of  diazobenzol  and  potassium 
hydrate.  This  substance  is  best  obtained  from  milk  containing 
tyrotoxicon  as  follows  :  The  filtered  milk,  which  is  acid  in  reaction, 
is  neutralized  with  sodium  carbonate,  agitated  with  an  equal  volume 
of  ether,  allowed  to  stand  in  a  stoppered  glass  cylinder  for  twenty- 
four  hours,  the  ether  removed,  and  allowed  to  evaporate  spontane- 
ously in  an   open  dish.      The   aqueous    residue    is  acidified  with 


324  CHEMISTRY  OF  THE  PTOMAINS. 

nitric  acid,  then  treated  with  an  equal  volume  of  a  saturated  solution 
of  potassium  hydrate,  and  the  whole  concentrated  on  a  water-bath 
(this  compound  is  not  decomposed  below  130°).  On  being  heated 
the  mixture  becomes  yellowish-brown,  and  emits  a  peculiar  aromatic 
odor.  On  cooling  the  tyrotoxicon  compound  forms  in  beautiful,  six- 
sided  plates  along  with  the  prisms  of  potassium  nitrate. 

With  equal  parts  of  sulphuric  and  carbolic  acids,  pure  tyrotoxicon 
gives  a  green  coloration,  but  in  whey  the  color  varies  from  yellow 
to  orange-red.  This  color  reaction  may  be  used  as  a  preliminary 
test  in  examining  milk  for  tyrotoxicon.  It  is  best  carried  out  as 
follows  :  Place  on  a  clean  porcelain  surface  two  or  three  drops  each 
of  pure  carbolic  and  sulphuric  acids.  Then  add  a  few  drops  of  the 
aqueous  solution  of  the  residue  left  after  the  spontaneous  evaporation 
of  the  ether.  If  tyrotoxicon  be  present,  a  yellow  to  orange-red 
coloration  will  be  produced.  This  test  is  to  be  regarded  only  as  a 
preliminary  one,  for  the  coloration  may  be  due  to  the  presence  of  a 
nitrate  or  nitrite,  or,  as  Huston  and  Weber  have  shown,  to  butyric 
acid.  The  tyrotoxicon  must  be  converted  into  the  potassium  com- 
pound and  purified  before  the  absence  of  nitrate  or  nitrite  can  be 
positively  demonstrated.  Moreover,  the  physiological  test  should 
always  be  made  in  testing  for  this  poison. 

With  platinum  chlorid  in  alcoholic  solution  tyrotoxicon  forms  a 
compound  which  explodes  with  great  violence  at  the  temperature  of 
the  water-bath.  This  also  corresponds  with  the  compound  of  plat- 
inum chlorid  and  diazobenzol.  It  should  be  borne  in  mind,  how- 
ever, that  organic  peroxids  may  behave  in  a  similar  manner. 

Pure  tyrotoxicon  is  insoluble  in  ether,  and  its  extraction  from 
alkaline  solutions  by  this  solvent  is  due  to  the  presence  of  foreign 
matter  with  which  the  poison  is  taken  up  by  the  ether. 

The  physiological  action  of  this  ptomain  has  been  discussed  in  a 
preceding  chapter. 

Mydalein  {/uudaUoi:,  putrid)  is  a  poisonous  base  obtained  in  1885 
from  putrefying  cadaveric  organs,  liver,  spleen,  etc.  (Brieger,  II.,  31, 
48).  Though  it  is  apparently  present  on  about  the  seventh  day,  it 
is  unobtainable  until  about  the  third  or  fourth  week.  The  method 
for  its  separation  from  the  accompanying  bases  is  given  under  saprin 
(page  278).  It  is  liable  to  occur  in  the  mercuric  chlorid  filtrate,  as 
well  as  in  the  precipitate,  inasmuch  as  the  double  salt  is  insoluble 
only  in  perfectly  absolute  alcohol.  In  order  to  purify  the  platino- 
chlorid  obtained  as  on  page  279,  it  is  repeatedly  recrystallized  from 
a  very  small  quantity  of  lukewarm  water.  This  base  has  not  been 
isolated  in  sufficient  quantity  to  permit  of  a  complete  determination 
of  its  composition.  It  is  probably  a  diamin,  containing  four  or  five 
carbon  atoms,  and  hence  is  nearly  related  to  some  of  the  diamins 
already  described. 


A  BASE.  325 

The  platinochlorid,  on  analysis,  gave:  Pt  =  38.74,  C  =  10.83, 
H  =  3.23.  It  crystallizes  in  small  needles,  and  is  extremely  soluble 
in  water. 

The  hydrochlorid  crystallizes  with  extreme  difficulty,  even  on 
standing  for  some  time  in  a  desiccator.  On  exposure  to  the  air  it 
rapidly  deliquesces. 

Mydalein  has  an  entirely  specific  action.  Small  quantities  injected 
into  guinea-pigs  or  rabbits  produce,  after  a  short  time,  a  moistening 
of  the  under  lip,  and  an  abundant  flow  of  secretion  from  the  nose 
and  eyes.  The  pupils  dilate  gradually  to  maximum  and  become 
reactionless ;  the  ear-vessels  become  strongly  injected,  and  the  body 
temperature  rises  1°  to  2°.  The  hairs  bristle,  and  the  animal  occa- 
sionally shudders.  Gradually  the  salivation  ceases,  the  respiration 
and  heart-action,  which  were  at  first  hastened,  now  decrease,  the 
temperature  falls,  the  ears  become  pale,  and  the  animal  finally  re- 
covers. During  the  action  of  the  poison  the  animal  shows  a  tend- 
ency to  sleep,  and  the  peristaltic  action  of  the  intestines  is  heightened. 
Larger  doses  (0.050  gram)  induce  an  exceedingly  violent  action  which 
invariably  results  in  the  death  of  the  animal.  On  post-mortem  the 
heart  is  found  to  be  stopped  in  diastole,  and  the  intestines  and  bladder 
are  contracted ;  otherwise  nothing  abnormal  is  observed. 

A  Toxic  Base. — From  human  livers  and  spleens  which  were  de- 
composing for  two  weeks  in  thorough  contact  with  air  there  was  iso- 
lated, besides  cadaverin  and  putrescin,  a  small  quantity  of  poisonous 
base  (Brieger,  II.,  29,  48).  The  mercuric  chlorid  precipitate  was  de- 
composed, and  the  hydrochlorids  were  precipitated  by  gold  chlorid 
(to  remove  cadaverin,  which  is  soluble),  and  the  aurochlorid  was 
then  changed  into  platinum  salt,  whereby  the  insoluble  putrescin 
platinochlorid  was  removed.  In  the  mother-liquor  from  the  putres- 
cin salt  an  easily  soluble  platinum  compound  was  detected  and 
found  to  contain  41.30  per  cent.  Pt.  It  crystallized  in  fine  needles. 
The  hydrochlorid  formed  small,  readily  deliquescent  needles,  and  did 
not  produce  a  precipitate  in  alcoholic  platinum  chlorid.  Injected 
into  guinea-pigs  and  rabbits  it  induced  an  exalted  peristalic  action 
of  the  intestines,  which  lasted  several  days  and  produced  in  the 
animals,  on  account  of  the  continuous  evacuations,  a  condition  of 
great  weakness.  No  disturbance  in  the  functions  of  the  other  organs 
was  observed. 

A  Base  was  isolated  from  decomposing  haddock  which  were  ex- 
posed for  five  days  during  summer  in  an  open  iron  vessel.  Brieger  (I., 
42)  found  in  the  aqueous  mercuric  chlorid  precipitate  (see  page  135) 
a  base  the  hydrochlorid  of  which  crystallized  in  well  formed,  small 
needles.  The  platinochlorid  likewise  crystallized  in  beautiful  needles 
and  gave,  on  analysis,  36.03  per  cent,  of  Pt;  7.81  per  cent,  of  N. 


326  CHEMISTRY  OF  THE  PTOMAInS. 

A  substance  of  muscarin-like  action  was  obtained  by  Brieger  (I., 
59)  from  putrefying  gelatin,  ten  days  at  35°,  though  in  insufficient 
quantity  to  permit  a  determination  of  its  character.  The  residue 
containing  this  substance  gave,  on  distillation  with  alkali,  only 
ammonia. 

A  Base  was  obtained  by  Bocklisch  (III.,  52,  53)  from  herring 
which  had  undergone  putrefaction  for  twelve  days.  It  was  found  in 
the  distillate,  together  with  trimethylamin  and  dimethylamin,  obtained 
by  distilling  the  mercuric  chlorid  filtrate,  after  the  removal  of  the 
mercury,  with  sodium  hydrate.  The  platinochlorid  was  easily  solu- 
ble, and  crystallized  in  large  thin  plates.  On  analysis  it  gave  : 
Pt  =  28.57,  C  =  22.34,  H  =  4.66.  The  hydrochlorid  was  easily 
soluble  in  water  and  in  absolute  alcohol,  and  besides  with  platinum 
gave  only  with  phosphomolybdic  acid  a  yellow  precipitate  which  was 
soluble  in  excess  and  with  ammonia  developed  an  immediate  blue  color. 
It  immediately  reduced  a  mixture  of  ferric  chlorid  and  potassium 
ferricyanid  with  formation  of  Berlin  blue ;  and  similarly  threw 
down  metallic  gold  from  solutions  of  gold  chlorid. 

From  poisonous  mussel,  Brieger  (III.,  79)  obtained  an  aurochlorid 
of  a  base  crystallizing  in  needles.  The  quantity  isolated  was  insuffi- 
cient for  analysis.  It  is  interesting  because  of  its  property  of  in- 
ducing salivation,  a  symptom  which  was  observed  by  Schmidtmann 
and  by  Crumpe  in  some  cases  of  mussel  poisoning. 

A  Base  was  obtained  by  Guareschi  and  Mosso  {Journ.  fur  prak- 
tische  Chem.,  28,  508)  from  fresh  beef,  in  the  alkaline  ether  extract 
obtained  by  Dragendorff's  method.  It  formed  a  yellowish  alkaline 
fluid,  of  unpleasant  odor,  and  after  a  time  gave  a  deposit  of  micro- 
scopic crystals.  The  hydrochlorid  gave  the  following  reactions  : 
Gold  chlorid,  yellow  crystalline  precipitate ;  platinum  chlorid,  pre- 
cipitate;  potassium  iodid  and  iodin  in  hydriodic  acid,  kermes-red 
precipitate ;  phosphotungstic  acid,  nothing ;  phosphomolybdic  acid, 
an  abundant  yellow  precipitate ;  tannic  acid,  heavy,  grayish  precipi- 
tate, same  with  Mayer's  reagent ;  picric  acid,  yellow  precipitate ; 
Marme's  reagent,  precipitate  soluble  in  excess  ;  potassium  bichromate, 
nothing ;  potassium  permanganate  and  sulphuric  acid,  violet  color ; 
potassium  ferricyanid  and  ferric  chlorid,  Prussian-blue  precipitate. 

By  giving  a  precipitate  with  tannin,  and  not  with  phosphotungstic 
acid,  it  resembles  neurin. 

Ch.  Gram  has  studied  the  decomposition  of  yeast  under  the  influ- 
ence of  an  infusion  of  hay.  The  yeast  was  allowed  to  putrefy  for 
fourteen  days,  and  was  then  treated  with  zinc  sulphate.  The  latter 
was  precipitated  by  barium  hydrate,  and  the  filtrate  after  the  removal 
of  the  barium  by  sulphuric  acid  was  evaporated  to  dryness  and  ex- 
tracted with  absolute  alcohol.     The  alcoholic  solution  was  evaporated, 


A  BASE.  327 

and  the  residue  again  extracted  with  alcohol.  The  extraction  residue 
was  taken  up  with  water,  and  again  subjected  to  the  above  treatment 
with  zinc  sulphate,  barium  hydrate,  etc. 

The  filtrate  was  poisonous  and  produced,  in  frogs,  paralysis  and 
stoppage  of  the  heart  in  diastole.  Addition  of  platinum  chlorid  and 
alcohol  precipitated  two  bases.  One  of  these,  although  possessing  a 
curara-like  action,  did  not  affect  the  heart.  When  its  solution  was 
heated  for  twenty-four  hours  on  a  water-bath  it  caused  general  paraly- 
sis and  stoppage  of  the  heart.  The  platinochlorid  contained  38.05 
per  cent,  of  platinum. 

The  other  base  also  possessed  a  slight  curara-like  action  and 
its  platinochlorid  gave,  on  analysis,  40.92  and  39.4  per  cent,  of 
platinum. 

Brieger  found  a  basic  substance  in  small  quantities  in  cultures  of 
the  staphylococcus  pyogenes  aureus  on  bouillon  and  beef-broth 
(II.,  74).  The  hydrochlorid  formed  groups  of  colorless  non-deli- 
quescent needles,  ^yith  platinum  chlorid  it  yielded  a  double  salt, 
crystallizing  in  needles,  and  containing  32.93  per  cent,  of  Pt.  For 
its  reactions,  see  Table  I. 

From  aqueous  as  well  as  alcoholic  extracts  of  cultures  of  staphylo- 
coccus aureus,  Leber  (1888)  isolated  a  crystalline  substance  which 
he  n&med  jMogosin.  The  composition  of  this  substance  is  not  known. 
It  does  not  seem  to  contain  nitrogen,  and  inasmuch  as  it  blackens 
silver  it  probably  contains  sulphur.  It  crystallizes  in  fine  needles 
which  are  soluble  in  ether  and  in  alcohol ;  difficultly  soluble  in  water. 
It  sublimes  in  needles.  Alkalis  precipitate  it  as  amorphous  yellow 
floccules  which  are  soluble  in  acid  and  then  can  be  recrystallized. 
With  potassium  ferricyanid  and  ferric  chlorid  it  yields  a  blue  color, 
and  with  potassio-mercuric,  cadmic,  and  bismuth  iodids  precipitates 
which  are  soluble  in  excess.  No  precipitate  is  produced  by  gold  or 
platinum  chlorid,  phosphotungstic,  or  molybdic,  tannic,  or  picric 
acids. 

A  small  quantity  applied  to  the  conjunctiva  produces  intense 
inflammation,  suppuration,  and  necrosis.  Introduced  into  the  anterior 
chamber  it  induces  intense  suppuration  and  keratitis.  The  substance 
is  entirely  distinct  from  the  base  obtained  by  Brieger  and  described 
above. 

A  Base — boiling-point  about  284° — was  obtained  by  Brieger  (II., 
61)  from  human  livers  and  spleens  which  were  putrefying  for  two  or 
three  weeks.  It  occurs  in  the  mercuric  chlorid  filtrate,  as  described 
under  saprin,  page  279,  together  with  some  mydalein,  trimethylamin, 
and  a  hydrocarbon.  The  filtrate,  after  the  mercury  is  removed  by 
hydrogen  sulphid,  is  evaporated  to  dryness,  and  finally  the  last  traces 
of  water  are  removed  in  a  vacuum.  The  residue  is  then  treated  with 
absolute  alcohol,  and  from  this   alcoholic  solution  the  mydalein  is 


328  CHEMISTRY  OF  THE  PTOMAINS. 

precipitated  by  the  addition  of  alcoholic  mercuric  chlorid.  The  tri- 
methylamin  is  separated  by  distillation  of  the  alkaline  filtrate,  pre- 
viously deprived  of  its  mercury  by  hydrogen  sulphid ;  while  the 
mother-liquor  yields  an  oily  mixture  of  hydrocarbons  and  bases. 
The  latter  were  separated  by  fractional  distillation,  w^hereby  only  one 
of  the  bases  was  obtained  in  sufficient  quantity  for  study.  It  boiled 
at  about  284°,  and  gave  with  hydrochloric  acid,  on  evaporation,  a 
salt  crystallizing  in  beautiful,  long  needles  which  were  very  easily 
soluble  in  perfectly  absolute  alcohol.  With  gold  chlorid  and  picric 
acid  it  gave  only  oily  products ;  with  ferric  chlorid  and  potassium 
ferricyanid,  an  intense  blue ;  with  platinum  chlorid,  an  extremely 
easily  soluble  double  salt  which  appeared  under  the  microscope  in 
the  form  of  very  fine  needles ;  while  from  alcohol-ether  the  double 
salt  slowly  separated  in  thin  plates  which  contained  30.36  per  cent, 
of  platinum.  The  free  base  showed  a  slight  fluorescence.  It  is 
not  poisonous  and,  according  to  Brieger,  is  probably  a  pyridin 
derivative. 

Other  non-poisonous  bases  were  present  in  very  small  quantity  in 
the  mother-liquor  described  above,  after  the  separation  of  the  oily 
mixture. 

Peptotoxin. — By  this  name  Brieger  (I.,  14-19)  designated  a 
poisonous  base  which  he  found  in  some  peptons,  and  hence  in  the 
digestion  of  fibrin ;  in  putrefying  albuminous  substances,  such  as 
fibrin,  casein,  brain,  liver,  and  muscles.  It  is  a  well  known  fact  that 
animal  tissues,  in  the  early  stages  of  putrefaction,  possess  strong  toxic 
properties,  even  before  the  decomposition  has  advanced  far  enough  to 
effect  a  marked  splitting  up  of  the  proteid  and  carbohydrate  mole- 
cules. Brieger  and  others  have  tried  to  seek  an  explanation  of  this 
toxicity  by  connecting  it  with  an  early  peptonization  of  the  proteids 
brought  about  by  the  action  of  enzymes  which  are  distributed 
throughout  the  tissues,  and  which  begin  their  activity  immediately 
after  death.  This  poison  has  not  been  definitely  isolated,  but  its 
general  properties  and  action  have  been  studied  by  Brieger,  who  pre- 
pared it  by  digesting  fibrin  for  twenty -four  hours  with  gastric  juice 
at  the  temperature  of  the  blood.  The  perfectly  fresh  pepton  thus 
obtained  was  evaporated  to  a  syrupy  residue,  and  this  was  then 
extracted  with  boiling  alcohol.  The  residue  left  on  evaporation  of 
the  alcoholic  solution  was  digested  for  some  time  with  amyl  alcohol 
which  on  subsequent  evaporation  gave  amorphous  brownish  masses. 
This  extract  was  then  purified  by  neutral  lead  acetate.  The  filtrate, 
after  the  removal  of  the  lead  by  hydrogen  sulphid,  was  repeatedly 
extracted  with  ether,  then  evaporated  to  dryness  and  extracted,  as 
before,  with  amyl  alcohol.  This  final  extract  was  evaporated  to 
drive  off  the  alcohol,  taken  up  with  water  and  filtered.  The  color- 
less aqueous  solution  thus  obtained  contains  the  poisonous  substance 


!iitepre< 
Insolub 
Iter,  on 
(ng  hec 
crystal! 
iluminc 
cipits 


lite  cur 
cipitj 


No  p 


rty-wh 
culeDt 
luble  in 

Ed  amo 
precipi 
illowls 
nine  p: 
tati 


hite  crj 
liible  in 


allow  1 

ite,  soli 

hot  wi 

No.  r 


fhite  gi 
cryst. 

tate,  ap 
slow 

rown  gi 
precip 


I  preeip 
(fine  ne 


Table  I. — Tabular  View  of  the  Reactions  of  Certain  Ptomatns. 


Ucil  j)rDalj>iliil(). 
YoUowIhIi  [)ro- 


YotlowiHli-wliito 


I,  HolUltiyiiiK 


Olcthylnmln 
Hydroctilorld, 


Yellow  precliil- 
I'rcclnltat^  cully 


[trlck-rcd  [irccljil- 
Oiiy  i.r«el|»Uiito, 


liiUoii.  AlcolioMo 

Urowii  oily  jm 
ulpllnto. 


tonio  ClilorUl  nud 

I'olatHUim  I 

oyniilil. 
rouuwlum  III 

lualo   uuil    Coii- 

coiitmlod      6ul- 


Boso  (p.  1 


Ycllowlsh-wliite 


:lpllate,dimciillly 
HOl.  In  Nir.O" 
)io  blue  coll 


•'Irsl  amorplioiis, 
Hillflowor  Htmpo. 


While  pi'cclpitiile. 


Yellowisli-TTjillo 


Prucipltutcorrcd 


lIORvy  yollowprc- 
olplUto. 

Wliltoproolpltato. 
Kormos-coloTcil 

Id. 

Imiiicdlato  llorlln 
bliio  proDlpltato. 

Poliisaluiii  bloUro- 

linrdlyorvHtallliio 
proQlpltn 


Yellow  needles. 

Dirty-white  pre- 
cipitate. 

Oily  preclpitnto, 
Hoaa  becomes 
cryBl-allinc. 


predpltatu.soh 
While  cryatalliE 


precipitate. 
Urown  prccipi- 


broad  needles. 


White  crysitalliiK 


While  precipilale, 


Brown  needles. 

Id. 

No  blue  color 
when  pure. 

Reddish-brown 
>rce[pitate  which 
ioOD  disappears. 


Uydrochlorid, 
C.H,.N,.2UCI. 


White  cryslalline 


Precipita 
slowly  ill  bt 
rill  yellow  ucedles 


CjHjNj.HCI. 


Oily  drops. 


Dirty-white  toIii. 


Cholin  Chlorid, 


Bctaiu  Chlorid, 


c,u„m^.uc\. 


I    Base  rroni  Cul- 
tures of  Typhoid 

BaclUtiB 
(Brieger,  11.  6! 


Dirty-white  doc- 
solSbll'iu'lxcess 


'S^, 


readily  soluble 
Yellow  needles. 


wbeu  sides  n 

rubbed  =  yell 

needles. 


Yellow  precipl-   'Whitoprcclpitat 


Yellowisli-whlio 


IIolTa's  Plomalii 


fhito  preclpitnto, 
Clonr  yellow 


CllltUTCJf  0 

S)aphylococ< 
pyogcTios  niir< 


Uydrochlorid. 
E  Heavy  while  pre- 


t  Yellowishpreclpi- 
tnte,  soluble  In 
NHiOH;  no  blue 


Yellow  needles. 


Plocculent  ycl- 


Oily  drops, 
hlicroscoplc 


slight  precipitate. 


A  precipitate. 
Brown   prcolpi- 


cipltnte. 

After  a  time  gives 
1  blue  coloration. 

Alcoholic  ZnCl, 


PEPTOTOXIN.  329 

which,  however,  can  only  with  extreme  difficulty  be  brought  to  crys- 
tallization in  vacuo. 

Salkowski  in  eight  digestion  experiments  with  fresh  fibrin  obtained 
a  poisonous  extract  in  but  one  case.  On  the  other  hand,  putrid 
fibrin  or  prolonged  digestion,  both  implying  bacterial  activity,  yield 
a  poisonous  product.  Peptic  digestion  of  serum-albumin,  egg- 
albumin,  and  meat  likewise  gave  negative  results.  In  view  of  these 
facts  as  well  as  its  presence  in  putrefying  proteids,  Salkowski  con- 
cludes that  a  peptotoxin  (in  the  sense  of  Brieger)  does  not  exist. 
That  poisonous  products  develop  in  meat  and  in  proteids  on  putre- 
faction is  well  established.  In  1891  Brieger  ascribed  a  proteid 
nature  to  peptotoxin,  excluding  it  from  the  bases.  Stadthagen  exam- 
ined normal  urine  for  peptotoxin,  but  failed  to  find  it. 

From  animals  with  extensive  burns  Kijanitzin  has  isolated,  by 
means  of  Brieger's  method  for  peptotoxin,  from  the  urine,  blood, 
and  especially  the  organs,  a  substance  resembling  peptotoxin  in 
chemical  and  physiological  behavior.  Atropin  antagonizes  its  action. 
The  clinical  symptoms  in  cases  of  extensive  burns  are  closely  allied 
to  those  observed  in  animals  when  a  part  or  whole  of  the  body  is 
varnished.  Rabbits  die  when  \—\  of  the  body-surface  is  varnished. 
Death  in  these  cases  has  been  explained  by  excessive  loss  of  heat  or 
by  lack  of  excretion  of  waste  products  of  the  skin.  Kijanitzin  holds 
that  the  varnishing  alters  the  chemical  products  of  cells  of  the  skin 
so  that  poisons  are  formed  and  carried  throughout  the  body  as  in 
skin-burns. 

Peptotoxin,  when  in  its  purest  condition,  as  shown  by  its  failure 
to  give  the  biuret  test,  possesses  a  neutral  reaction.  Its  behavior 
to  Millon's  reagent  is  quite  characteristic ;  it  gives  a  white  pre- 
cipitate which  on  boiling  becomes  intensely  red.  Because  of  this 
reaction,  Brieger  is  inclined  to  regard  this  substance  as  a  hydroxyl 
or  an  amido  derivative  of  benzol.  The  ptomain  can  be  extracted 
from  acid  as  well  as  alkaline  solution  by  amyl  alcohol — more  diffi- 
cultly in  the  cold  than  on  heating.  It  is  absolutely  insoluble  in 
ether,  benzol,  and  chloroform ;  very  soluble  in  water.  It  is  not 
destroyed  by  boiling,  by  passing  hydrogen  sulphid,  or  by  strong 
alkalis  ;  but  is  destroyed,  however,  when  the  putrefaction  lasts  longer 
than  eight  days.     For  its  behavior  to  reagents,  see  Table  I. 

Various  observers  have  shown  that  pepton  possesses  a  toxic  action, 
and  some  have  been  led  to  regard  this  toxicity  as  not  due  to  the 
pepton  itself,  but  rather  to  the  presence  of  this  or  some  other 
ptomain.  At  least  Brieger  found  one  specimen  of  dry  Witte's  pepton 
to  be  perfectly  harmless  ;  whereas,  the  fresh  pepton  formed  by  fibrin 
digestion  possessed  strong  toxic  powers.  Moreover,  this  non-poison- 
ous pepton  when  exposed  to  the  action  of  gastric  juice  was  found  to 
yield  the  poisonous  substance.  It  is  indeed  probable  that  pepto- 
toxin is  not  a  distinct  compound  but  rather  a  mixture  as  in  the  case 


330 


CHEMISTRY   OF  THE  PTOMAINS. 


of  the  toxalbumins.     The  poisonous  nature  of  proteids  and  the  physio- 
logical action  of  this  base  will  be  described  later. 

Pyocyanin,  Cj^Hj^NgO,  is  the  coloring  matter  of  blue  pus,  and  is 
produced  by  the  action  of  the  bacillus  pyocyaneus.  It  was  isolated 
by  Ledderhose  (1887)  and  is  said  to  be  an  anthracene  derivative. 
On  contact  with  the  air  it  is  oxidized  to  pyoxanthose,  a  yellow  sub- 
stance. According  to  Kunz,  it  contains  nitrogen  and  sulphur.  The 
picrate  is  of  a  dark  reddish-brown  color ;  the  platinum  salt  is  black, 
and  sometimes  is  obtained  as  glittering  fine  golden  needles. 

Table  of  Ptomains. 


Formula. 

Name. 

Discoverer. 

Physiological 
Action.  1 

C  HgN 

Methylamin. 

Bockliscli. 

Non-poisonou8. 

CJH7N 

Dimethylamin. 

Brieger. 

<(            (( 

C3H9N 

Trimethylamin. 

Dessaignes. 

u               n 

C,  H5  N 

Spermin  (?). 

Kunz. 

11            It 

C,  H^N" 

Ethylamin. 

Hesse. 

<<            (( 

C,  HnN 

Diethylamin. 

Bocklisch. 

((            t( 

Ce  H15N 

Triethylamin. 

Brieger. 

((            (( 

CaHgN 

Propylamin. 

11 

C,  H,,N 

Butylamin. 

Gautier  &  Mourgues 

Poisonous  (?). 

C5  HiiN  (?) 

Tetanotoxin. 

Brieger. 

Poisonous. 

Ci  H13N 

Amylamin. 

Hesse. 

<( 

Ce  II15N 

Hexylamin. 

It 

(< 

Ct  HiiN 

Di-hydrolutidin. 

Gautier  &  Mourgues 

(( 

Cs  HnN 

Collidin  (?). 

Nencki. 

Cs  HuN 

Pyridin  base  (?). 

0.  de  Coninck. 

Cs  H13N 

Hvdrocollidin  (?). 

Gautier  &  Etard. 

Poisonous. 

C9  H„N 

Parvolin  (?). 

(1       ((      (( 

C10H15N 

Unnamed. 

Guareschi  &  Mosso. 

Poisonous. 

CioH.jN 

Pyridin  base  (?). 

0.  de  Coninck. 

C3.HS1N 

Unnamed. 

Del^zinier. 

' 

C,  H3  N, 

Ethylidenediamin  (?). 

Brieger. 

Poisonous. 

C3  He  N, 

Anthracin. 

Hoffa  (1889). 

Cs  Hs  N, 

Trimethylenediamin  (?). 

Brieger. 

Poisonous. 

C,  Hi,N, 

Putreacin. 

(( 

Not  very  poisonous. 

C5  H14N2 

Cadaverin. 

<< 

((       ti          <( 

C5  H„N, 

Neuridin. 

<< 

Non-poisonous. 

C5  H,,N, 

Saprin. 

li 

a                u 

Ce  H,eN, 

Hexamethylenediamin. 

Garcia. 

C,  H,oN, 

Unnamed. 

Morin. 

Non-poisonous. 

CioH^eNj  (?) 

Susotoxin. 

Novy. 

Poisonous. 

C,  H,  N, 

Methyl  guanidin. 

Brieger. 

(< 

*-19'^27^J 

Morrhuin. 

Gautier  &  Mourgues 

Diuretic,  etc. 

^13"2oN4 

Unnamed. 

Oser. 

^lltljgiN  J 

(( 

Gautier  &  Etard. 

CasHs^N, 

Asellin. 

Gautier  &  Mourgues 

Poisonous. 

C5  H13N  0 

Neurin. 

Brieger. 

K 

Cs  Ht,N  0 

Mydin. 

(1 

Non-poisonous. 

C5  H„N  0, 

^-amido-valerianic  acid. 

E.  &  H.  Salkowski. 

<i             (( 

C,  H.^N  0, 

Cholin. 

Brieger. 

Poisonous. 

Ce  H13N  0, 

Mydatoxin. 

(( 

11 

^  Only  those  bases  are  here  denoted  as  poisonous  which  possess  a  decided  toxicity. 


MINATIONS. 

abner's  Inaugural  Dissertation. 


Brouardel  and  Boutmy. 

Baumert 
(Liebermann). 

Otto. 

Cadaver. 
In  water  18  months. 

Parts  of 

putrefying  goose 

and  cadaver. 

anby  hydro- 
lie  acid. 

Ether  (acid). 

Yellow,  amorphous; 
taste  sharp,  bitter. 

Petroleum-ether 

Residue 

Alkaline. 

.\Ikaline,  volatile 
liquid  ;  odor  that 
of  urine  of  mice. 

(alkaline). 
Bright  yellow  oil. 

White  precipitate. 
Violet  precipitate. 

Same  as  colchicin; 
soluble  in  alcohol. 
Same  as  colchicin. 

A  precipitate, 

colehicin=0. 

Same  as  colchicin. 

Gold  Cb. 

A  precipitate. 
Id. 

Kermes-brown 
precipitate. 
Orange-red. 

sium 

Phosphe 

Acid. 

Sulphune  cold, 
ish-violet. 

Sulphur 
audP 
Bichn 

Same  as  colchicin, 
blue  with  NH^OH. 

Yellow  color. 

Colorless  ;  on 
warming,  violet. 

No  odor  of  butyric 
acid. 

Dark-yellow  color, 

concentrated  HNO3 

=carmine  red, 

which  with  water 

gives  yellow. 



llydroc' 

Acid. 

Mercuri 

Cherry-red  on 
heating. 

Abundant 
precipitate. 

Colorless. 

A  precipitate. 

Same  as  colchicin. 

A  precipitate, 

colchicin=0. 

Zeisel's  reaction=0, 

colchicin,  green. 

Ferric  salts=blue. 

Millon's  reagent 

showed  presence  of 

pepton. 

Non-poisonous. 

Chlor 
Chlorin 

Potassium  ferri- 

cyanid  is  reduced, 

HjS04  +  BaH.^0„  = 

brick  red  ;  on 

warming,  violet. 

Non-poisonous. 

Physioli,  produces 
Actioi  of  heart, 
is,  death. 

NoH-poisorwus. 

Intensely 
poisonous. 

Table  II. — PtomaIns  in  Toxicological  Examinations. 
Note. — The  Greater  Part  op  thi-s  Table  has  been  Taken  Direct  from  Grabner's  Inaugurai.  Dissertation. 


Rorsch 
and  Fassbender. 

Schwanert. 

Liebermann. 

Zuelzer  and 
Sonnenschein. 

T.  Gelder. 

Brouardel  and  Boutmy, 

Baumert 

(Liebermann). 

Otto. 

Liver  of  decompoK- 
ing  cadaver  and  of 
fresh  liver  of  ox. 

Decomposing 

liver,  kidney,  and 

stomach. 

Putrefying 

stomach  and 

contents. 

Muscle  macerated 
in  water. 

Liver,  kidney, 
stomacn  and  intes- 
tine of  an  ex- 
humed arsenic- 
containing  body. 

Cadaver. 
Death  by  asphyxia. 

Cadaver. 
Death  by  hydro- 
cyanic acid. 

Cadaver. 
In  water  18  months 

Parts  of 

putrefying  goose 

and  cadaver. 

ainined. 

Solvent. 
Kesidue. 

Ether  (acid  and 

alkaline). 

Amorphous,  not 

bitter. 

Ether  (alkaline). 

Liquid,  volatile ; 
repulsive  taste. 

Ether  (acid  and 

alkaline). 
Resinous,  brown- 
ish, soluble  in 
water ;  acid  taste. 

Ether  (alkaline). 

Greasy  brownish 
mass,  with  crystals. 

Ether  (acid). 

Yellow,  amorphous 
taste  sharp,  bitter. 

Petroleum-ether 

(alkaline). 
Bright  yellow  oil. 

Brown  extract. 

Alkaline. 

Alkaline,  volatile 
liquid;  odor  that 
of  urine  of  mice. 

White  precipitate. 

Gradual 

cloudiness. 

Bluiah-yellow 

precipitate. 

Dirty-yellow  aix- 

sided  stars. 
Clear  brown  pre- 
cipitate. 
Splendid  blue, 
later  green. 
Yellow  precipitate ; 
with  NH4OH, 
blue. 
Dirty  brownish- 
yellow,  unchanged 
on  warming. 
Reddish-brown, 
then  grass-green. 

Yellow. 

Deliquescent 

white  needles. 

White  crystalline 

precipitate. 
Dirty  wnite  pre- 
cipitate. 

White  precipitate. 

White  precipitate. 

Yellow  crystalline 

precipitate. 
Brownish-yellow 

precipitate. 

Kerraes-brown 

precipitate. 

White  precipitate. 
Brown  precipitate. 

Id. 

Id. 

White  precipitate. 
Y'ellow  precipitate. 

Kermes-brown  precipitate. 

White  precipitate. 
Violet  precipitate. 

Same  as  colchicin; 
soluble  in  alcohol. 
Same  as  colchicin. 

A  precipitate, 

colchicin=0. 

Same  as  colchicin. 

A  precipitate. 

Yellowish  precipi- 
tate. 
Yellowish-brown 
precipitate. 

Yellow  to  dark 
brown. 

Kermes-brown 
precipitate. 
Orange-red. 

siinn  lodid. 
Frohde's  Reagent. 

Phosphomolybdic 

Sulphuric  Acid. 

Sulpliiiric  Acid 
and  Potassium 
Hicliromate. 

Yellow  precipitate, 
on  warming  green  ; 
with  Nli^OIl.blue. 

Yellow  precipitate. 

Colorless,  then 

slight  reddish-violet 

color. 

Heavy  flocculent 
precipitate. 

Yellow  precipitate. 

On  warming 
becomes  yellow. 

White  precipitate. 
On  warming,  violet. 

Same  as  colchicin, 
blue  with  NH.1OH. 

Yellow  color. 

In  the  cold, 
browuish-violet. 

Colorless ;  on 
warming,  violet. 

No  odor  of  butyric 
acid. 

Yellow  spots  on 
evaporation. 

Id. 

Golden-yellow. 

Dark-yellow  color, 

concentrated  HNO3 

=carmine  red, 

which  with  water 

gives  yellow. 

Cherry-red  on 
beating. 

Abundant 
precipitate. 

Colorless. 

Acid. 

White  cloudiness, 

Curdy  white  pre- 
cipitate. 

White  precipitate. 
Id. 
Id. 

Y'ellow  precipitate. 

Same  as  colchicin. 

A  precipitate, 

colchicin=0. 

Zeisel's  reaction=0, 

colchicin,  green. 

Ferric  8alts=blue. 

Millon's  reagent 

showed  presence  of 

pepton. 

Nou-poisonoua, 

Clilorid. 

Heavy  white  pre- 
cipitate. 



Potassium  ferri- 

cyanid  is  reduced, 

H^SO^  +BaH^03  = 

brick  red;  on 

warming,  violet. 

Non-poiflonou8. 

When  fed 

to  pigeons  no 

etfect. 

Causes  mydriasis 
and  increase  in  the 
rate  of  heart-beat. 

precipitate.    Iodic 

acid  is  reduced. 

Silver  nitrate.white 

precipitate  with 

reduction  of 

silver.     Ferric 

chlorid  =0. 

In  frogs,  produces 
slowing  of  heart, 
paralysis,  death. 

Non-poisonous. 

Intensely 

Action. 

poisonous. 

Table  III. — Reactions  of  Selmi's  Ptomains. 


TABLE  OF  PTOMAINS. 


331 


Table  of  Ptomains. — Continued. 


Formula. 

Name. 

Discoverer. 

Physiological 
Action.' 

Ce  H„N  0, 

Unnamed. 

Brieger,  1888. 

Non-poisonous. 

Q  H15N  0, 

Mytilotoxin. 

Brieger. 

Poisonous. 

G,  H„N  0, 

Gadinin. 

II 

(1 

Cj  Hi,N  O2 

Typhotoxin. 

II 

II 

Ct  H17N  0, 

Unnamed. 

(1 

II 

Ci,Hi,N,0 

Pyocyanin. 

Ledderhose. 

Non-poisonous. 

C5  H13N  O3 

Betain. 

Brieger. 

i(           II 

C5  H15N  0, 

Muscarin. 

(1 

Poisonoua. 

C9  HisN  O3 

Morrhuic  acid. 

Gautier&  Mourgues 

C5  HijNjO^ 

Unnamed. 

Pouchet. 

Poisonous. 

^13^30^  2^4 

Tetanin. 

Brieger. 

II 

Ci,H,oN,0, 

Unnamed. 

Guareschi. 

CieH^NA 

<( 

Lepierre. 

Poisonous. 

Ct  Hi8N,0« 

Pouchet. 

Tyrotoxicon. 

Vaughan. 

Mydalein. 

Brieger. 

Spasmotoxin. 

11 

A  diamin  {?). 

II 

Peptotoxin. 

(1 

Phlogosin. 

Leber. 

Inflammatory. 

'Only  those  bases  are  here  denoted  as  poisonous  which  possess  a  decided  toxicity. 
For  Griffiths'  bases,  see  page  261 . 


CHAPTER   XV. 

CHEMISTEY   OF   THE    LEUCOMAiNS. 

Under  this  head  are  classed  those  basic  substances  which  exist 
preformed  in  the  proteids  or  other  constituents  of  the  body  or  are 
produced  as  products  of  tissue  metabolism.  The  proteid  molecule  is 
usually  the  parent  of  the  ptomains  and  also  of  the  leucomains.  The 
distinction  between  these  two  groups  is  therefore  necessarily  imper- 
fect and  unsatisfactory.  In  the  one  case  bacteria  are  at  work  and 
split  up  the  proteid,  giving  rise  to  ptomains.  The  latter  are  essen- 
tially cleavage  products  and  the  differences  which  exist  among  these 
bases  are  to  be  sought  largely  in  the  peculiar  composition  of  the  pro- 
teid acted  upon  and  perhaps  to  less  extent  in  the  individual  germ 
which  is  the  active  agent  of  destruction.  The  fact  that  few,  if  any, 
of  the  ptomains  can  be  shown  to  be  synthetic  products  indicates  that 
the  bacteria  or  their  enzymes  act  largely  as  hydrolytic  agents. 

Obviously,  the  living  animal  cell,  through  its  enzyme,  may  produce 
the  same  or  similar  basic  products  which,  however,  being  non-bacterial 
in  origin  are  designated  as  leucomains.  Furthermore,  these  basic  sub- 
stances may  be  liberated  by  the  direct  action  of  hydrolytic  agents 
other  than  enzymes.  Thus,  to  illustrate,  the  nuclein  molecule  when 
acted  upon  by  bacteria  yields  the  xanthin  bases.  The  latter  are 
formed  in  the  living  animal  and  plant  in  tissue  metabolism  and  also 
result  by  the  action  of  dilute  mineral  acids.  The  hexon  bases,  while 
they  have  not  been  as  yet  found  to  be  bacterial  products,  will  un- 
doubtedly be  shown  to  be  such,  for  the  reason  that  they  are  formed 
by  the  enzymes  of  yeast,  of  germinating  plants,  of  pancreas  and  of 
the  liver,  and  because  they  are  easily  split  off  from  proteids  by  the 
action  of  acids.  Again  lecithin,  the  parent  substance  of  cholin,  is 
widely  distributed  in  plants  and  animals.  It  yields  cholin  when 
acted  upon  by  bacteria  or  by  acids,  or  when  it  undergoes  metabolism 
in  the  living  plant  or  animal.  The  same  is  true  for  the  well  known 
ptomains,  putrescin  and  cadaverin,  which  according  to  Lawrow  are 
formed,  undoubtedly  from  lysin  and  arginin,  in  the  sterile  autodiges- 
tion  of  pigs'  stomachs. 

The  first  attempt  at  the  systematic  study  and  generalization  of 
these  basic  substances  was  made  by  Gautier,  who  applied  to  them  the 
name  leucomains,  a  term  derived  from  the  Greek,  ke'jycona,  signify- 
ing white  of  eggs.  Under  this  name  he  included  all  those  basic 
substances  which  are  formed  in  animal  tissues  during  life,  in  contra- 
distinction to  the  ptomains  or  basic  products  of  putrefaction.     Many 

332 


LEUCOMAINS.  333 

of  these  bases  exist  preformed  in  plants  or  can  be  readily  prepared 
from  them. 

Thus  vegetable  tissues  are  known  to  contain  not  only  what  are 
ordinarily  designated  as  ptomains,  such  as  cholin,  but  also  leuco- 
mains,  of  the  hexon  and  purin  group.  Indeed,  in  this  latter  group 
must  be  placed,  on  account  of  their  relation  to  xanthin,  those  well 
defined  alkaloidal  bases,  caffein,  theophyllin  and  theobromin.  Not 
only  are  the  representatives  of  these  two  divisions  of  basic  substances 
common  to  both  kingdoms,  but  their  parent  bodies,  lecithin,  nuclein, 
and  proteids  occur  in  both,  and  hence  give  rise  to  the  same  bases  on 
decomposition. 

Gautier  believed  that  the  leucomains  are  being  formed  continu- 
ously and  incessantly  in  the  animal  tissues  side  by  side  with  the  for- 
mation of  urea  and  carbonic  acid,  and  at  the  expense  of  the  nitrog- 
enous elements.  It  is  quite  probable,  as  Kossel  has  pointed  out, 
that  some  of  these  products  are  in  themselves  antecedents  of  end- 
products  of  metabolism.  This  is  unquestionably  true  of  the  amido 
group,  which  exists  in  the  adenin  and  guanin  molecules,  and  through 
vital  or  putrefactive  processes  is  split  off,  giving  rise  to  ammonia 
which  in  turn  serves  to  form  urea.  More  than  that,  the  researches 
of  the  past  few  years  have  shown  that  the  purin  and  hexon  bases, 
while  primary  cleavage  bodies,  are  largely  broken  down  into  more 
simple  metabolic  products.  Thus,  the  uric  acid  which  is  formed  in 
certain  organs  in  the  body  of  mammals  is  chiefly,  if  not  wholly,  de- 
rived by  the  oxidation  of  the  purin  bases  or  of  their  antecedent. 
The  kidneys,  muscles  and  even  the  liver  itself  may  destroy  uric  acid, 
and  for  that  reason  the  amount  of  uric  acid  excreted  does  not  indicate 
the  amount  that  is  formed  (Wiener).  The  cleavage  of  uric  acid  into 
allantoin,  oxalic  acid  and  urea  is  well  known,  and  Minkowski  has 
recently  shown  that  allantoin  is  also  formed  after  the  administration 
of  purin  bases,  as  well  as  of  thymus  glands. 

Bouchard  endeavored  to  explain  the  presence  of  leucomains  in 
the  urine,  by  supposing  that  they  were  originally  formed  in  the 
intestinal  tract,  from  which  they  were  absorbed  into  the  system,  to 
be  subsequently  eliminated  by  the  kidneys.  This  view  has  also  been 
brought  forward  by  Schar  (1886)  who  held  that  these  bases  may  be 
formed  by  putrefactive  changes  in  the  intestinal  tract,  in  which  case 
they  are  absorbed  into  the  circulatory  system,  whence  they  may  be 
partly  eliminated  by  the  kidneys  or  may  be  partly  deposited  in  the 
tissues  themselves. 

The  views  of  Bouchard  and  Schar  have,  to  a  certain  extent,  been 
confirmed  by  the  investigations  of  Udranszky  and  Baumann,  who 
showed  that  the  well  known  ptomains  cadaverin  and  putrescin  occur 
in  the  urine  in  cystinuria,  and  are  formed  by  putrefactive  changes 
induced  in  the  intestinal  tract,  probably  by  specific  microorganisms. 
Under  this  same  head  fall   the  observations  of  Wolkow  and  Bau- 


334  CHEMISTRY   OF  THE  LEUCOMAINS. 

mann,  that  alkapton  is  produced  from  tyrosin  by  similar  changes  in 
the  intestines.  The  production  of  intestinal  products,  their  absorp- 
tion and  excretion  by  the  kidneys,  is  likewise  seen  in  such  well 
known  compounds  as  phenol,  indol,  skatol,  etc.  The  origin  of  the 
true  leucomains  cannot,  however,  be  accounted  for  in  this  manner, 
for  they  are  indissolubly  connected  with  the  metabolism  of  the  cell 
itself,  and  are,  therefore,  formed  in  the  tissues  and  organs  proper.  As 
such  they  are  endogenous  whereas  the  intestinal  products  are  chiefly 
the  result  of  bacterial  action  and  are  therefore  of  exogenous  origin. 

The  leucomains  have  been  credited  by  many  as  playing  the  chiet 
r6le  in  auto-intoxications.  It  should  be  noted,  however,  that  most 
of  the  basic  bodies  which  have  been  studied  up  to  the  present  are 
far  from  being  poisonous.  The  antecedents  of  these  bases,  how- 
ever, such  as  the  nucleinic  acids  and  more  especially  the  histons  and 
protamins  exert  a  pronounced  toxic  action  and  hence  may  play  a 
most  important  part  in  auto-intoxications.  Of  course,  it  must  be 
admitted  that  there  may  be  generated  within  the  body  toxic  leuco- 
mains which,  as  yet  unknown,  normally  undergo  prompt  destruction. 
The  fact  that  amino  derivatives  of  purin  and  pyrimidin  are  highly 
toxic  is  deserving  of  attention. 

The  known  sources  of  leucomains  are  the  nucleins  of  the  nuclei  and 
the  proteids  of  the  protoplasm  of  the  cells.  The  former  gives  rise 
to  the  purin  and  pyrimidin  bases  whereas  the  latter  generate  the  hexon 
bases.  In  addition  to  these  there  is  a  fairly  large  number  of  substances, 
as  yet  but  poorly  studied,  which  remain  to  be  accounted  for.  Cholin 
and  allied  bodies  may  be  looked  upon  as  leucomains  derived  from 
lecithin.     They  have  been  described  in  the  preceding  chapter. 

The  leucomains  may  be  divided  into  the  following  groups  :  (1) 
the  purin  or  uric  acid  group  ;  (2)  the  pyrimidin  group  ;  (3)  the  hexon 
bases  ;  (4)  the  oreatinin  group. 

The  first  of  these  divisions  contains  a  number  of  well  known  bases 
which  are  derivatives  of  purin  and  for  that  reason  are  closely  allied 
to  uric  acid. 

Purin,  C6H4N4. 

AdENIN,  C5H5N5. 

HyPOXANTHIN,  C5H4N4O. 

GuANm,  CjHsNsO. 

Methyl  gtjanin,  C6H7N5O  (Epiguanin). 

XaNTHIN,  C5H4N4O2. 

(Uric  ACID,  CsH^N.Oj). 

1-METHYL  XANTHIN,  C6H6N4O2. 

3-METHYL  XANTHIN,  CgHgN^O,. 

7-METHYL  XANTHIN,  C6H6N4O2  (Heteroxanthin). 

1-7  Di-METHYL  XANTHIN,  C7H8N4O2  (Paraxanthin). 

1-3  Di-METHYL  XANTHIN,  CyHgN^Oj  (Theophyllin). 

3-7  DI-METHYL  XANTHIN,  C^HgN^Oj  (Theobromin). 

1-3-7  TRi -METHYL  XANTHIN,  C8H10N4OJ  (Caffein). 

CaRNIN,  C7H8N4O3. 

Carnosin,  C9H]4N40g. 

Cytobin,  CjiHaoNie04. 


PURIN  BASES.  335 

The  pyrimidin  group  may  be  considered  as  antecedents  or  per- 
haps more  correctly  as  cleavage  products  of  the  purin  bodies.  It 
is  represented  by  the  following  : 

Pyrimidin,  C^H^N,.  A  body,  C8HeN404. 

Ubacil,  C^H^NjO,.  Episakkin,  C^HgNaO.     (?) 

5-METHYii  Uracil,  CjHjNjOj  (Thymin).  Pseudoxanthin,  C4H5N5O.     (?) 

The  hexon  bases  proper  are  only  three  in  number.  To  these  may 
be  appended  a  lower  homologue,  two  amines  and  a  related  acid 
which  may  be  looked  upon  as  cleavage  products  of  the  hexons.  The 
third  group  then  will  include  : 

AbGININ,      CgHi4]Sr402.  PYRROIilDIN   CARBONIC  AciD,    CgHgNO,. 

HisTiDiN,    CgHgNgOj.  Gerontin,  CgHi^Nj. 

Lysin,         CeHi^NjOj.  Spermin,  C2H5N  (?) 

Obnithin,  CgHj^NjO,.  Methyl  Quinolin,  CjoHgN. 

The  members  of  the  fourth  group  have  all  been  discovered  by 
Gautier  and  by  him  are  regarded  as  allied  to  creatin  and  creatinin. 
These  two  substances,  especially  the  latter,  have  been  hitherto 
regarded  as  strongly  basic  in  character,  but  Salkowski  has  shown 
that  creatinin,  when  perfectly  pure,  possesses  little  or  no  alkaline 
reaction,  and,  moreover,  does  not  combine  with  acids.  In  structure 
and  properties  it  approximates  the  pyrimidin  bodies.  The  bases  in 
this  group  are  : 

(Creatinin,  C^HjNsO). 

(Creatin,  C^HjNgOj). 

Cruso-creatinin,        CjHgNiO. 

Xantho-creatinin,     CjH^oN^O. 

Amphi-creatin,  CgHigN^O^. 

Base,  CuHj^NioOj. 

Base,;  Ci^H^sNiiOs. 

Besides  these  two  general  classes  of  leucomains,  there  may  be 
made  a  fifth  class  of  undetermined  leucomains,  embracing  those 
bases  which  have  been  observed,  but  studied  more  or  less  incom- 
pletely, in  the  various  physiological  secretions  of  the  body. 

PURIN   BASES. 

The  substances  included  under  this  head  were  formerly  spoken  of 
as  xanthin  bases  because  of  their  relation  to  xanthin,  the  earliest 
known  member  of  the  group.  Subsequently,  the  term  nuclein  bases 
was  employed  to  designate  the  original  four  xanthin  bases,  inasmuch 
as  they  are  derived  from  the  nucleins.  The  latter  on  decomposition 
with  acids  yields  one  or  more  of  these  basic  products.  Kossel  and 
Kriiger,  in  1894,  suggested  a  new  term  based  upon  the  fact  that  the 
xanthin  bases  like  uric  acid  contain  alloxan  and  urea  groups.  They 
designated  therefore  these  as  alloxuric  bases.  On  the  other  hand  the 
expression  "alloxuric  bodies"  was  made  to  include  uric  acid  as  well 
as  the  xanthin  bases. 


336  CHEMISTBY  OF  THE  LEUCOMAINS. 

The  studies  of  Fischer  in  1884  on  uric  acid  led  him  to  consider 
this  substance  as  a  derivative  of  a  hypothetical  body,  CgH^N^,  to 
which  he  gave  the  name  purin.  The  remarkable  researches  of  this 
investigator  during  the  past  few  years  have  shown  that  the  xanthin 
bases  are  actually  related  to  uric  acid  and  that  they  also  are  deriva- 
tives of  purin.  This  parent  substance  of  uric  acid  and  of  the 
xanthin  bases  was  not  prepared  until  1898,  although  as  stated,  its 
existence  was  pointed  out  fifteen  years  before.  The  term  purin 
bodies  therefore  includes  purin  and  all  of  its  derivatives.  The  purin 
bodies  formed  in  the  animal  organism  and  appearing  in  the  urine  are 
uric  acid  and  the  several  xanthin  bases.  The  expression  purin 
bases  implies  the  same  as  the  older  terms  xanthin,  nuclein  or  allox- 
uric  bases. 

Before  considering  the  individual  purin  bases  it  is  desirable  to 
point  out  the  main  facts  bearing  upon  the  constitution  or  structure 
of  the  members  of  this  group.  A  thorough  understanding  of  the 
structural  relationship  of  these  several  bodies  is  necessary  in  order 
to  appreciate  the  role  which  they  play  in  tissue  metabolism.  The 
waste  product  uric  acid  differs  from  xanthin  in  that  it  contains  one 
atom  of  oxygen  more  than  does  the  latter.  Xanthin  in  turn  differs 
in  the  same  way  from  hypoxanthin.  For  some  time  the  belief  has 
prevailed  that  the  nuclei  of  cells  contain  the  physiologically  impor- 
tant xanthin  bases.  These  bases  do  not  exist  free  but  rather  in  com- 
bination with  proteid  substances  forming  the  so-called  nucleinic 
acids,  or  the  more  complex  nucleins.  When  the  nucleins  undergo 
metabolism  the  purin  bases  are  set  free,  suffer  more  or  less  oxida- 
tion and  leave  the  body  partly  as  uric  acid  and  partly  as  cleavage 
products  such  as  the  pyrimidin  bodies,  creatinin,  allantoin,  urea, 
oxalic  acid,  etc.  These  successive  changes  may  be  rendered  appar- 
ent by  comparing  the  formulae  of  these  bodies — 

PUKIN,  CgH^N^. 

Hypoxanthin,     CgH^N^O. 
Xanthin,  CgH^N^Oj. 

Uric  Acid,  CgH^N^Og. 

Although  these  four  substances  form  a  homologous  group  it  does 
not  follow  that  uric  acid  can  be  readily  prepared  by  oxidation  from 
either  of  the  preceding  or  that  vice  versa  it  can  be  reduced  to  these 
bases.  The  statement  made  by  Strecker  that  hypoxanthin  by  the 
action  of  fuming  nitric  acid  yields  a  nitro-product,  which  on  reduc- 
tion gives  xanthin,  was  taken  to  indicate  that  hypoxanthin  possessed 
a  constitution  similar  to  that  of  xanthin.  Kossel,  however,  showed 
in  1882  that  this  conversion  of  hypoxanthin  into  xanthin  did  not 
take  place,  and  in  this  he  was  confirmed  by  Fischer  in  1884.  In 
the  same  way  the  observation  of  Rheineck,  reported  by  Strecker, 
that  uric  acid  is  converted  by  sodium  amalgam  into  xanthin,  and  this 
still  further  reduced  to  hypoxanthin,  has  been  shown  to  be  incorrect 


PURIN  BASES.  337 

by  Fischer,  who  failed  to  change  xanthin  into  uric  acid  or  uric  acid 
into  xanthin.  While  therefore  uric  acid,  xanthin,  and  hypoxanthin 
contain  three,  two,  and  one  atoms  of  oxygen,  respectively,  and  are 
structurally  closely  allied,  nevertheless  thus  far  it  has  not  been  pos- 
sible to  convert  directly  any  one  of  those  compounds  into  the  others. 

Only  very  recently  Sundwik  has  attempted  the  reduction  of  uric 
acid  by  boiling  it  with  sodium  hydrate  and  chloroform.  By  this 
procedure  he  believes  that  he  has  prepared  xanthin  and  hypoxanthin, 
but  a  confirmation  of  these  results  is  necessary. 

By  electrolytic  reduction  of  uric  acid  Tafel  ^  obtained  a  substance 
which  he  has  designated  as  puron,  CgH^N^Oj.  This  body  is  neither 
acid  nor  basic  in  character  and  is  therefore  a  fully  saturated  com- 
pound. It  readily  changes  into  an  unsaturated  isomer — iso-puron.  A 
tetrahydrouric  acid,  C^HgN^Oj,  was  also  obtained  which  with  baryta 
gave  a  —  /9  diaraino-propionic  acid,  C3HgN202 ;  and  with  nitrous 
acid  a  base  C^H^NjOg. 

The  classical  investigations  of  Fischer,  however,  have  shown  that 
indirectly  uric  acid  may  be  changed  to  xanthin  or  to  hypoxanthin 
and  these  bases  in  turn  by  a  similar  indirect  process  can  be  converted 
into  uric  acid.  Thus,  on  treating  1-3  di-methyl  uric  acid  with  phos- 
phorus pentachlorid  Fischer  obtained  chlor-theophyllin,  which  on 
reduction  with  hydriodic  acid  gave  theophyllin  or  di-methyl  xan- 
thin. By  introducing  a  methyl  group  into  the  latter  caffein  or  tri- 
methyl  xanthin  results.  In  a  similar  way  caffein  may  be  prepared 
from  1-3-7  tri-methyl  uric  acid.  Inasmuch  as  the  di-  and  tri-methyl 
uric  acids  can  be  prepared  artificially  it  follows  that  the  complete 
synthesis  of  theophyllin  and  of  caffein  was  effected  by  this  trans- 
formation. 

Subsequently  (1897)  Fischer  converted  potassium  urate  by  means 
of  phosphorus  oxychlorid  into  an  oxy-di-chlor  purin  and  this  into 
tri-chlor  purin.  The  latter  by  a  series  of  transformations  which  it 
is  unnecessary  to  detail  was  changed  into  xanthin,  hypoxanthin, 
adenin,  guanin,  heteroxanthin  and  paraxanthin.  With  the  exception 
of  methyl  xanthin  all  of  the  known  purin  bases  found  in  the  urine 
have  been  therefore  not  only  made  from  uric  acid  but  their  complete 
synthesis  has  also  been  effected. 

The  starting  point  in  the  consideration  of  the  structure  of  the 
purin  bodies  is  uric  acid,  which  is  not  only  the  earliest  known,  but 
also  the  most  easily  obtainable  and  best  studied  member  of  this 
group.  The  structure  of  uric  acid  as  given  by  Medicus  in  1875 
was  verified  and  fully  established  by  the  studies  of  Fischer  in  1884. 
On  the  other  hand,  these  early  investigations  of  Fischer  on  the  xan- 
thin bases  led  him  to  ascribe  to  these  somewhat  different  formulae 
than  did  Medicus.  When,  however,  Fischer  in  1895  succeeded  in 
converting  di-methyl  uric  acid  into  chlor-theophyllin  it  was  evident 

^Berkhte,  34,  258,  1181  (1901). 
22 


338 


CHEMISTRY  OF  THE  LEUCOMAINS. 


that  the  xanthin  bases  possessed  essentially  the  same  structure  as  uric 
acid.  This  fact  was  rendered  still  more  evident  when  hydroxy-caf- 
fein  not  only  was  recognized  to  be  a  tri-methyl  uric  acid,  but  on 
treatment  with  methyl  iodid  was  shown  to  form  the  tetra-methyl 
uric  acid.  Fischer  therefore  abandoned  the  formulae  of  caffein  and 
xanthin  which  he  proposed  in  1883  and  accepted  those  which  Medi- 
cus  had  previously  suggested. 

The  formula  which  Medicus  suggested  and  which  shows  best  the 
various  decompositions  of  uric  acid  is  that  given  below  (1).  The  tauto- 
meric form  (2)  has  a  great  deal  in  its  favor,  but  is  not  used.  Similar 
tautomeric  formulae  can  be  deduced  for  purin,  trichlor  purin,  adenin 
and  other  bases. 


(1) 
HN— CO 


OC 


HN— C— NH 


— NH 
O 


HO. 


(2) 
=C.OH 


C. 

N— C— N 


•NH 
OH 


Ukic  Acid. 


The  group  C^N^  in  the  above  formulae  is  designated  as  the  purin 
nucleus  and  from  it  purin  itself,  CgH^N^,  is  easily  derived.  The 
nomenclature  of  purin  bodies  is  based  upon  the  order  of  substitution 
in  the  purin  nucleus,  C^N^.  Nine  positions  are  possible  as  shown  in 
the  second  formula  below  : 


:h 


N=CI 
HC     C— NH 


CH 

II 
N— C— N 

PUEIN. 


N3-C,- 


Uric  acid  becomes  2-6-8  tri-oxypurin.  By  the  introduction  of  a 
single  methyl  group  into  uric  acid  a  methyl  uric  acid  results.  On 
examining  the  formula  of  uric  acid  it  will  be  seen  that  theoretically 
four  methyl  uric  acids  are  possible.  Strange  to  say  there  are  present 
six  known  mono-methyl  uric  acids ;  in  other  words  there  are  two 
more  than  are  theoretically  possible. 


CH3.N-CO 
OC     C— NH 


HN— CO 
OC    C— 


o 


NH 

!0 


HN— CO 

C— N- 


OC 


CH3 


HN— CO 

OC     C— NH 


HN— C— NH         CH3N— G-NH  HN— C— NH  HN— C— N.CH3 

1-Methyl  Uric  Acid.     3-Methyl  Uric  Acid.   7-Methtl  Uric  Acid.    9-Methyl  Uric  Acid. 

By  substituting  two  methyl  groups  into  uric  acid  a  di-methyl  uric 
acid  results.  Six  di-methyl  uric  acids  are  possible  and  all  are 
known,  of  the  tri-methyl  uric  acids  four  are  possible  and  are  like- 


PUBIN  BASES.  339 

wise   all   known.     One   tetra-methyl  uric  acid  is  possible  and   is 
known  but  in  addition  an  isomer  exists  in  raethoxy-caffein. 

Xanthin. — The  successful  synthesis  of  xanthin  by  Fischer  (1897) 
has  shown  that  this  interesting  base  is  2-6-di-oxypurin  and  as  such 
possesses  the  formula  originally  deduced  by  Medicus.  A  considera- 
tion of  the  purin  formula  will  show  that  three  di-oxypurins  are 
possible.  Xanthin  therefore  has  two  isomers,  6-8  and  2-8  dioxy- 
purins,  the  latter  of  which  Is  as  yet  unknown  although  methyl  de- 
rivatives have  been  prepared. 

HN— CO  HN— CO  HN— CO 

OC     C— NH  NH,.C    C— NH  NH,.C    C— N.CH3 

II    in  II    CH  II    I    CH 

I    II  II     II  II    II    II 

HN— C— N  N— C— N  N— C— N 

Xanthin.  Guanin.  Epigdanin  or 

7-Methyl  Qcanin. 

The  methyl  derivatives  of  xanthin  are  of  great  interest  inasmuch 
as  they  make  up  the  greater  part  of  the  natural  purin  bases.  By 
replacing  a  hydrogen  atom  in  either  one  of  the  three  imido  groups 
1,  3,  or  7,  in  xanthin  by  a  methyl  group  a  mono-methyl  xanthin 
results.  All  three  of  the  possible  mono-methyl  xanthins  are  known. 
The  1 -methyl  xanthin  was  discovered  in  urine  by  Kriiger  and  Salo- 
mon in  1897,  but  as  yet  it  has  not  been  synthesized.  Heteroxanthin, 
which  is  7-methyl  xanthin  (Kriiger  and  Salomon  1895),  was  likewise 
isolated  from  urine  by  Salomon  in  1884.  The  3-methyl  xanthin  has 
been  isolated  from  the  urine  after  administration  of  large  doses  of 
caffein. 

CH3.N— CO  HN— CO  HN— CO 

00    C— NH  OC    C— NH  OC    C— N.CH, 

I      II      I  I      II      I  I      II     J. 

CH  CH  CH 

HN— C— N  CH3.N— C— N  HN— O— N 

1-Mkthyl  Xanthin.         3-Methtl  Xanthin.  Hetkrox.4Nthin  or 

V-Mkthyl  Xanthin. 

Only  three  di-methyl  xanthins  are  possible  and  these  have  been 
known  to  exist  in  nature  for  many  years.  They  are  theobromin,  the 
active  principle  of  theobroma  cacao  ;  theophyllin,  which  exists  in  tea, 
and  paraxanthin  which  occurs  in  the  urine.  The  relationship  which 
exists  between  these  three  isomers  is  readily  seen  from  the  formulae, 


CH3.N— CO  HN— CO 

OC     C— N.CHj  OC     C— N. 

I  II  L  I  i 


CH3 

H 
HN— C— N  CH,.N— C— N 


Theophyllin  or  Paraxanthin  or  Theobromin  or 

1-8  Di-MBTUYL  Xanthin.    1-7  Di-methyl  Xanthin.        3-7  Di-methyl  Xanthin. 


340  CHEMISTRY  OF  THE  LEUCOMAINS. 

Only  one  tri-methyl  xanthin  is  possible  and  that  derivative  is  the 
active  principle  of  tea  and  coffee — caffein. 

If  the  tautomeric  formula  of  xanthin,  with  the  imido  group  trans- 
ferred from  position  7  to  9,  is  considered  it  will  be  seen  that  additional 
derivatives  of  xanthin  are  possible,  such  as  a  9-methyl  xanthin,  1-9  and 
3-9  di-methyl  xanthins  and  a  1.3.9-tri-methyl  xanthin.  The  lat- 
ter is  an  isomer  of  caffein  but  unlike  the  latter  possesses  but  little 
action  upon  the  muscle  (Schmiedeberg,  Berichte,  34,  2556). 

In  1881  Fischer  showed  that  caffein  possessed  the  formula  given 
below.  In  the  following  year  he  succeeded  in  changing  xanthin  into 
theobromin  which  in  turn  can  be  easily  methylated  to  caffein.  In 
1897  Fischer  successfully  synthesized  all  of  the  natural  xanthins 
except  1 -methyl  xanthin.  The  origin  of  the  di-methyl  and  mono- 
methyl  xanthins  was  not  understood  until  but  recently.  Unlike 
xanthin,  these  derivatives  when  found  in  the  urine  cannot  be  traced  to 
tissue  metabolism,  but  on  the  contrary  they  result  by  the  splitting 
off  of  one  or  more  methyl  groups  from  the  caffein  or  theobromin 
which  is  ingested.  Thus,  when  very  large  doses  of  caffein  are  given 
to  a  dog  all  three  methyl  groups  are  attacked  at  once.  The  one  in 
position  7  is  the  least  firm  and  hence  disappears  quite  readily.  Het- 
eroxanthin  or  7-methyl  xanthin  is  therefore  present  in  minimal 
amounts  and  for  the  same  reason  theophyllin  or  1-3  di-methyl  xan- 
thin is  found  in  larger  amounts  than  paraxanthin  or  theobromin.  It 
is  of  singular  interest  to  note  that  caffein  in  the  body  yields  not 
only  paraxanthin  but  also  theophyllin  and  theobromin  which 
have  been  known  heretofore  to  exist  only  in  the  vegetable  kingdom. 
The  purin  bases  which  result  from  the  cleavage  of  caffein  in  the 
body  vary  according  to  the  animal  used.  Thus,  while  feeding  caf- 
fein to  a  dog  yields  chiefly  theophyllin  (1-3)  and  3-methyl  xanthin, 
in  a  rabbit  it  yields  chiefly  paraxanthin  (1-7),  1-methyl  xanthin  and 
heteroxanthin  (7). 


CH,N— I 


OC 


-N.CHs 

m 


CHs-N— C— N 
Caffein. 


Guanin. — The  fact  that  guanin  is  readily  changed  into  xanthin 
shows  that  the  two  bases  are  closely  related.  Indeed,  the  relation  is 
the  same  as  that  between  adenin  and  hypoxanthin.  By  decomposi- 
tion with  concentrated  hydrochloric  acid  Wulff  obtained  the  same 
products  as  are  given  by  xanthin.  On  oxidation,  however,  with 
chlorin  neither  Strecker  nor  Fischer  were  able  to  obtain  alloxan 
and  urea,  but  instead  obtained  parabanic  acid,  guanidin,  and  car- 
bonic acid.  A  guanidin  residue  is  contained  in  the  molecule  in 
place  of  the  urea  residue  in  xanthin.     A  striking  difference  between 


PUEIN  BASES.  341 

xanthin  and  guanin  is  seen  in  the  difficulty  of  preparing  alkyl  deriva- 
tives of  guanin,  whereas  with  xanthin  the  result  is  easily  obtained. 
Fischer  and  Reese  failed  in  preparing  alkyl  derivatives,  and  Wulff 
succeeded  only  with  ethyl  guanin.  Recently,  however,  Fischer  has 
succeeded  in  effecting  the  synthesis  of  two  methyl  derivatives  and  of 
guanin  itself.  The  structural  formulae,  thus  deduced,  for  guanin 
and  epiguanin  are  given  on  p.  339.  It  will  be  seen  that  guanin  is 
2-amino-6-oxypurin,  while  epiguanin  is  7-methyl  guanin. 

Two  of  the  five  possible  isomers  of  guanin  have  been  prepared 
synthetically  by  Fischer.  One  of  these,  6-amino-2-oxypurin,  very 
closely  resembles  guanin,  so  much  so  that  it  can  be  easily  mistaken 
for  this  base. 

Hypoxanthin. — This  is  6-oxypurin  and  inasmuch  as  it  can  be 
readily  prepared  from  adenin  by  the  action  of  nitrous  acid,  it  follows 
that  the  latter  is  6-aminopurin.  Of  the  four  possible  formulae  for 
adenin  Fischer  selects  the  one  given  below.  Two  isomers  of  adenin 
are  possible. 

HN— CO  N=C.NH, 

Hc   c— NH  :hc  c— nh 

II    II   ('^  II    II   ^^ 

N— C— N  N— C— N. 

Htpoxahthin.  Adenin. 

It  should  be  remembered  that  there  are  isomers  of  both  of  these 
bases  and  that  tautomeric  forms  may  exist  as  in  the  case  of  uric  acid. 

The  relation  of  adenin  and  hypoxanthin  to  uric  acid  and  xanthin 
was  clearly  shown  by  Kriiger  in  1891  and  1893,  although  the  struc- 
ture of  these  bases  was  not  fixed  by  Fischer  until  1897.  Thus,  the 
bromin  derivatives  of  adenin  and  hypoxanthin  correspond  to  those  of 
xanthin,  guanin  and  caffein,  and  on  oxidation  with  chlorin  yield  alloxan 
and  urea.  The  bromin  derivatives  of  hypoxanthin,  adenin,  xanthin, 
guanin,  and  caffein  result  by  the  substitution  of  bromin  in  the  CH 
group  of  xanthin,  i.  e.,  in  position  8.  The  analogy  to  the  other 
members  of  the  uric  acid  group  is  seen  from  the  following  equations  : 


C,U;^P,  +  O  +  HP  =  CJI,^P,  +  CON,H,. 

Ubic  Acid. 

C.H^Np,  +  20  +  up  =  C^H^Np,  +  CON^H,. 

Xanthin. 

C^H.Np  +  30  +  up  =  C.H^Np,  +  CON^H,. 

Hypoxanthin. 

C,H,Np  +  30  +  up  =  C3H2N2O3  +  C.NH.N^H,  -{-  CO,. 

Guanin.  Pababanic  Acid.  Guanidin. 

C^H^N  +  30  +  2H,0  =  C^H^NjO,  +  CON^H,  +  NH3.  (?) 

Adenin. 


342  CHEMISTRY  OF  THE  LEUCOMAINS. 

The  equations  for  adenin  and  hypoxanthin  are  not  fully  estab- 
lished, but  the  bromin  compounds  are  decomposed  into  alloxan.  In 
addition  to  this  urea  and  oxalic  acid  are  formed,  both  of  which  may 
be  derived  from  alloxan  by  decomposition,  though  some  of  the  urea 
may  be  independent  of  the  breaking  down  of  the  alloxan  group. 

Again,  adenin  and  hypoxanthin  on  decomposition  with  concen- 
trated hydrochloric  acid  yield  the  same  products  (glycocoll,  ammonia, 
formic  and  carbonic  acids)  as  xanthin,  guanin,  etc.  This  decompo- 
sition is  best  seen  from  the  following  equations  : 

C,H,NP3  +  5Hp  =  3NH3  +  C^H.NO^  +  300^. 

Uric  Acid. 

C,H,Np,  +  mp  =  3NH,  +  C,H,NO,  +  2CO,  +  CHp,. 

Xanthin. 

•      C^H^Np  +  7H,0  =  4NH3  +  C^H^NO,  +  200^  +  CH,0,. 

Guanin. 

C^H.Np  +  7Hp  =  3NH3  +  C^HgNp  +  CO^  +  SCHp^. 

Hypoxanthin. 

C,H,N,  +  mp  =  4NH3  +  C^H^NO^  +  CO,  +  2CHA. 

Adenin. 

Uric  acid  has  no  CH  group,  hence  does  not  yield  formic  acid, 
whereas  xanthin  and  guanin  have  each  one  CH  group  and  yield  one 
molecule  of  formic  acid.  Hypoxanthin  and  adenin  therefore  have 
two  CH  groups. 

Furthermore,  uric  acid  contains  three  CO  groups  and  yields  three 
CO2  molecules,  whereas  xanthin  and  guanin  have  two  CO  groups 
and  yield  two  COg  molecules.  Hence  hypoxanthin  has  but  one  CO 
group,  since  it  yields  but  one  COg  molecule.  The  corresponding  car- 
bon atom  in  adenin  is  likewise  split  off  as  carbonic  acid. 

The  changes  which  the  purin  bodies  undergo  in  the  animal  econ- 
omy have  been  the  subject  of  numerous  investigations.  As  is  well 
known,  in  birds  the  greater  part  of  the  waste  nitrogen  passes  out  in 
the  form  of  uric  acid.  This  large  amount  of  uric  acid  is  derived 
from  two  sources  :  (1)  in  small  part  from  the  nuclein  metabolism  of 
the  tissue ;  (2)  chiefly  by  synthetic  transformation  of  simple  nitrog- 
enous substances.  Thus,  after  the  administration  of  urea  or  of 
ammonium  salts  of  organic  acids  birds  eliminate  uric  acid.  This 
synthesis,  according  to  Minkowski,  takes  place  within  the  liver,  since 
after  the  removal  of  that  organ  the  change  does  not  take  place. 
Instead  the  administered  nitrogen  is  excreted  as  ammonium  salts 
while  the  amount  of  uric  acid  falls  to  a  minimum  corresponding  to 
that  formed  by  actual  nuclein  metabolism. 

In  mammals  on  the  other  hand  the  purin  bodies  carry  off  only  a 
very  small  part  of  the  waste  nitrogen.  The  chief  if  not  the  sole 
source  of  the  uric  acid  is  tissue  metabolism,  since  the  recent  studies 


PURIN  B.LSES.  343 

of  Minkowski  (1898)  render  it  very  doubtful  that  a  uric  acid  syn- 
thesis occurs  in  mammals  in  a  manner  similar  to  that  in  birds.  In 
dogs  the  administration  of  urea,  ammonium  lactate  and  allantoin  is 
not  followed  by  excretion  of  uric  acid.  jS^evertheless  it  is  obvious  that 
purin  compounds  are  synthesized  in  the  body  out  of  the  food  sup- 
plied. The  growing  cell  can  undoubtedly  build  up  the  purin  molecule 
out  of  simple  material  and  is  in  no  wise  restricted  to  the  preformed 
nucleins  or  purins  in  the  food.  The  amount  thus  synthesized  in 
mammals  may  be  considered  as  not  in  excess  of  that  necessary  for 
the  purpose  of  the  cells  and  hence  the  synthetic  elimination  of  uric 
acid  is  not  recognized.  In  birds  on  the  other  hand  it  is  conceivable  that 
the  pronounced  synthetic  power  of  the  liver  is  directed  not  at  a 
mere  elimination  of  uric  acid  but  rather  at  the  formation  of  ante- 
cedents (purins  ?)  which  are  utilizable  by  the  cells  and  stored  up 
as  nuclein.  The  supply  of  such  antecedents  being  in  great  excess  of 
the  actual  needs  of  the  economy  leads  to  their  oxidation  and  as  a 
result  we  have  increased  uric  acid  elimination  which  from  this  stand- 
point would  correspond  to  that  of  urea.  The  greater  part  of  the 
latter  is  believed  to  result  from  the  breaking  down  of  the  circulat- 
ing proteids  when  these  are  in  excess  of  the  needs  of  the  tissues. 

When  nucleinic  acid  is  fed  to  mammals  the  amount  of  uric  acid 
eliminated  is  increased.  The  same  is  true  as  Taylor  and  others 
ha>e  shown  after  a  diet  of  sweet-breads  which  are  rich  in  nucleins. 
When,  however,  the  actual  amount  of  uric  acid  eliminated  is  com- 
pared mth  the  amount  of  nuclein  present  it  is  evident  that  by  far 
the  greater  part  of  the  purin  bodies  is  broken  up  and  disappears. 
Two  explanations  are  offered  on  this  point.  In  the  first  place  it  is 
possible  that  the  larger  part  of  the  purin  bodies  in  nuclein  is  oxi- 
dized direct  into  urea  and  only  a  small  part  goes  to  form  uric  acid. 
Or,  the  second  possibility  is  that  the  greater  part  is  oxidized  into 
uric  acid ;  this  in  turn  is  largely  broken  down  into  urea,  and  only  a 
small  portion  escaping  this  cleavage  appears  in  the  urine. 

It  is  well  known  that  uric  acid  when  fed  to  mammals  is  broken 
down  and  excreted  as  urea,  allantoin  (Salkowski),  oxalic  acid, 
glycocoll  and  other  products.  If  the  amount  of  uric  acid  fed  is 
excessive  allantoin  can  be  detected  in  the  urine,  otherwise  the  cleav- 
age is  carried  farther,  as  is  seen  in  the  fact  that  no  allantoin  can  then 
be  isolated.  The  studies  of  Wiener  (1899)  have  shown  that  certain 
organs  in  the  body  when  crushed  may  destroy  while  others  appar- 
ently make  uric  acid.  Moreover  the  action  of  a  given  organ  was 
found  to  vary  with  the  species  of  the  animal.  Thus,  the  liver,  spleen 
and  thymus  of  beef  were  found  to  make  uric  acid  whereas  the  liver 
of  dog  and  hog  actually  destroyed  this  substance.  Again,  the  kidney 
and  muscles  of  the  beef  and  horse  were  shown  likewise  to  destroy 
uric  acid  whereas  the  kidney  of  a  dog  did  not.  In  these  experiments 
the  antecedent  of  uric  acid  is  undoubtedly  nuclein  since  hypoxanthin 


344  CHEMISTRY  OF  THE  LEUCOMAINS. 

by  the  action  of  the  crushed  liver  is  changed  to  uric  acid.  In  beef- 
liver,  however,  Wiener  obtained  an  alcohol-soluble  antecedent  which 
was  not  a  purin.  It  is  very  doubtful  as  to  whether  uric  acid  can 
thus  be  synthesized  from  urea,  glycocoU  and  similar  compounds. 
One  fact  is  evident  and  that  is  that  uric  acid  and  hence  other  purin 
bodies  are  broken  down  in  the  body  and  that  this  change  takes  place 
especially  in  certain  organs. 

The  amount  of  uric  acid  actually  carried  to  the  kidneys  to  be 
eliminated  (a)  therefore  will  be  the  algebraic  sum  of  that  formed  in 
nuclein  metabolism  {x)  (tissue  and  food),  that  formed  by  synthesis 
[y),  and  that  destroyed  in  the  body  (2).     Thus, 

X  -^  y  —  z  =  a. 

The  actual  amount  of  uric  acid  eliminated,  however,  may  be  con- 
siderably less  than  that  represented  by  a  since  the  kidney  may  be 
incapable  of  effecting  the  complete  excretion.  In  which  case  de- 
posits of  uric  acid  will  occur  in  the  joints,  as  in  rheumatism  ;  and  in 
the  kidneys  as  in  ad  en  in  poisoning  and  in  the  kidney  infarcts  of  the 
new-born  (Spiegelberg). 

The  purin  bases  may  be  looked  upon  as  undergoing  changes 
similar  to  those  of  uric  acid.  Hypoxanthin  apparently  is  the  only 
member  of  the  group  which  when  fed  to  birds,  dogs  or  man  (Min- 
kowski) is  eliminated  as  uric  acid  or  as  its  cleavage  product  allantoin. 
Moreover  the  crushed  beef  liver  is  capable  of  effecting  this  conversion 
into  uric  acid  (Wiener).  Adenin  on  the  other  hand  in  dogs  does 
not  give  rise  to  uric  acid  or  to  allantoin.  A  small  amount  appears 
in  the  urine  unchanged  (Kossel,  Minkowski)  the  remainder  being 
undoubtedly  broken  up  into  simple  products.  It  is  significant  that 
the  thymus  gland  which  is  rich  in  an  adenin-producing  nuclein 
should  increase  the  amount  of  uric  acid  and  even  give  rise  to  allan- 
toin, whereas  adenin  itself  is  without  this  effect.  It  may  be  that  the 
adenin  while  still  in  a  nascent  condition,  as  it  were,  is  more  readily 
oxidized  than  when  definitely  formed.  At  all  events  its  antecedent 
behaves  quite  differently. 

According  to  Kriiger  and  Schmid  ^  the  purin  bases  when  admin- 
istered to  man  are  largely  eliminated  as  uric  acid.  Thus,  62.3  per 
cent,  of  the  hypoxanthin  given  was  excreted  as  uric  acid  and  only 
0.15-0.25  per  cent,  as  purin  bases.  Adenin  gave  a  similar  result, 
40.7-41.2  per  cent,  appearing  as  uric  acid  and  2.6-3.7  per  cent, 
being  unchanged.  With  xanthin  10  per  cent,  formed  uric  acid  and 
1  per  cent,  was  eliminated  unchanged.  Guanin  perhaps  also  yields 
uric  acid  but  this  is  not  definitely  settled. 

Nothing  definite  can  be  said  regarding  the  fate  of  xanthin  and 
guanin  in  the  animal  body.     It  has  been  shown  in  pancreatic  and 

'Zeite.  physiol.  Chem.,  34,  549,  1902. 


PURIN  BASES.  346 

yeast  auto-digestions  that  hypoxanthin  and  xanthin  are  readily  de- 
stroyed by  the  action  of  the  enzyme  whereas  adenin  and  guanin  resist. 
The  xanthin  derivatives  however  have  been  studied  carefully  and 
the  results  are  extremely  interesting.  Thus,  the  caffein  and  theo- 
bromin  of  the  food  are  progressively  broken  up  into  simpler  xanthin 
bodies  and  a  large  part  of  the  xanthin  molecule  is  completely  de- 
stroyed. The  cleavage  of  cafPein  will  be  described  in  detail  later  but 
in  this  connection  it  is  desirable  to  point  out  that  most  of  the  purin 
bases  found  in  the  urine  owe  their  origin  not  to  direct  tissue  met- 
abolism but  rather  to  the  cleavage  of  caffein  and  similar  bases 
introduced  with  the  food.  Inasmuch  as  the  increase  of  purin  bases 
is  not  proportional  to  the  amount  of  caffein  ingested  it  is  evident 
that  considerable  of  the  latter  is  destroyed.  Moreover  it  may  be 
considered  as  an  established  fact  that  caffein  does  not  increase  the 
amount  of  uric  acid  although  the  use  of  coffee  according  to  Taylor 
is  followed  by  a  marked  increase  in  the  elimination  of  this  product. 

The  pharmacological  action  of  the  purin  derivatives  has  been  re- 
cently (1901)  investigated  by  Schmiedeberg.  Owing  to  the  extremely 
slight  solubility  of  some  of  these  bases  it  follows  that  their  effects 
are  but  slight.  In  general  the  well  known  effects  of  caffein  are  re- 
produced, although  obviously  differences  exist.  The  action  of  caffein 
is  directed  upon  the  central  nervous  system,  the  muscles  and  the 
kidneys.  The  effects  on  the  former  are  seen  in  the  increased  reflex 
irritability  which,  as  in  the  case  of  strychnin,  may  lead  to  complete 
tetanus  and  even  paralysis.  The  muscles  contract  more  easily  and 
more  effectively  and  with  large  doses  they  become  permanently  con- 
tracted, passing  into  a  condition  of  coagulation  like  that  caused  by 
heat  and  cold  (see  Cushny's  Pharmacology).  The  action  on  the  kid- 
ney is  seen  in  the  marked  diuresis. 

Purin  exerts  a  slower  coagulation  effect  upon  muscles  and  causes 
increased  reflex  irritability.  The  resemblance  to  caffein  is  even  more 
marked  with  methyl  purin. 

Hypoxanthin  was  found  to  have  no  action  upon  muscle,  but  the 
effect  upon  the  nervous  system  was  seen  in  the  increased  irritability 
and  tetanus.  The  di-methyl  hypoxanthin  had  a  slight  action  upon 
the  muscles  and  also  tetanized.  On  the  other  hand  8-oxypurin,  the 
isomer  of  hypoxanthin,  did  coagulate  muscle,  but  showed  no  tetanic 
action,  whereas  its  di-methyl  derivative  showed  both  effects  and  was 
about  as  active  as  theobromin. 

Xanthin  agrees  in  its  action  fully  with  the  above  8-oxypurin ; 
that  is  it  causes  muscular  rigor  and  general  paralysis,  but  no 
increased  irritability.  The  alkyl  derivatives  of  xanthin  cause 
greater  rigor  of  the  muscles  and  otherwise  act  like  caffein  and 
theobromin.  Of  the  di-methyl  xanthins  theobromin  showed  less 
action  on  muscles  than  theophylliu,  while  paraxanthin  was  the  most 
active. 


346  CHEMISTRY  OF  THE  LEUCOMAINS. 

The  diuretic  action  of  the  purin  bodies  apparently  parallels  the 
effect  upon  muscles.  Thus,  paraxanthin  is  more  diuretic  than  theo- 
phyllin  while  theobromin  is  less  effective  than  the  latter.  The  in- 
creased reflex  irritability,  as  with  ammonia  compounds,  is  due  to  the 
nitrogen  groups,  whereas  the  action  upon  muscle  is  peculiar  to  the 
purin  molecule. 

Adenin,  unlike  the  other  purin  bases,  has  been  shown  by  Minkow- 
ski to  be  a  violent  poison.  The  profound  effects  induced,  which 
will  be  described  later,  are  apparently  due  to  the  presence  of  the 
amino  group.  Steudel  has  also  observed  a  marked  toxicity  follow- 
ing the  introduction  of  the  amino  group  into  pyrimidin. 


I.   The  Purin  Bases. 

Purin,  CgH^N^,  was  successfully  prepared  by  Fischer^  in  1898. 
It  is  of  interest  because  it  is  the  prototype  of  the  group  to  which  it 
lends  the  name.  Although  as  yet  it  has  not  been  met  with  in  the 
body  or  among  the  cleavage  products  of  proteids,  it  is  not  unlikely 
but  that  improved  methods  will  reveal  the  presence  of  purin  and  its 
methyl  derivatives.  A  brief  description  of  its  properties  which 
clearly  place  it  in  the  same  series  with  hypoxanthin,  xanthin  and 
uric  acid,  will  therefore  be  appropriate. 

Purin  forms  colorless  microscopic  needles  often  united  in  spherical 
aggregates.  The  melting  point  is  211-212°.  It  is  very  easily  sol- 
uble in  cold  water  and  the  solutions  do  not  react  toward  litmus  or 
curcuma.  It  is  also  very  easily  soluble  in  warm  alcohol,  from  which 
it  slowly  crystallizes  in  small  densely  felted  needles.  It  is  more 
difficultly  soluble  in  acetic  ether  and  in  aceton,  and  especially  so  in 
ether  or  in  chloroform.     It  forms  salts  with  acids  as  well  as  with  bases. 

The  nitrate,  CgH^N^.HNOg ,  is  very  easily  soluble  in  hot  water ; 
rather  difficultly  so  in  hot  alcohol.  It  crystallizes  in  roundish 
aggregates.  On  warming  with  dilute  nitric  acid  it  becomes  yellow. 
It  melts  at  205°  with  decomposition.  The  chlorid,  iodid  and  sul- 
phate are  extremely  soluble  in  water. 

The  picrate  is  difficultly  soluble,  requiring  about  twenty  parts  of 
boiling  water.    It  forms  yellow  glistening  needles  which  melt  at  208°. 

The  platinochlorid  forms,  upon  the  addition  of  platinum  chlorid 
to  fairly  concentrated  solutions  of  the  chlorid,  as  fine  yellow  needles 
which  readily  dissolve  on  heating.  Gold  chlorid  gives  an  oily  or 
resinous  precipitate,  which  in  time  forms  a  solid  yellow,  granular 
mass  which  is  also  readily  soluble  on  heating. 

The  sodium  salt  is  very  soluble  in  water,  difficultly  so  in  concen- 
trated sodium  hydrate  from  which  it  crystallizes  in  bundles  of  fine 
needles.     The  potassium  and  barium  salts  are  likewise  very  soluble. 

^Berichte,  31,  2550;  32,  493. 


ADENIN.  347 

With  ammoniacal  silver  solution  it  gives  at  once  a  colorless 
amorphous  precipitate,  which  is  not  blackened  by  boiling  or  by 
light.  Neutral  silver  nitrate  produces  a  white  precipitate  which  is 
soluble  in  hot  dilute  nitric  acid  and  separates  out  on  cooling  as  a 
white  granular  or  crystalline  powder.  Ammoniacal  zinc  solutions 
precipitate  purin,  especially  on  heating,  in  a  fine  pulverulent  form. 
Mercuric  chlorid  gives  an  amorphous  precipitate  which  on  boiling 
becomes  crystalline.  Phosphotungstic  acid  produces  an  extremely 
fine  precipitate,  imparting  a  milky  appearance  to  the  fluid.  Tannin 
produces  a  colorless  flocculent  deposit,  while  bismuth  iodid  in  acid 
solution  yields  a  red  granular  precipitate.  Potassium  iodid,  ferro- 
cyanid  and  Nessler's  solution  produce  no  precipitate. 

With  bromin  it  yields  a  beautiful  yellowish-red,  crystalline  mass, 
which  dissolves  on  heating  and  recrystallizes  on  cooling.  It  is  very 
resistant  to  oxidation  and  with  nitric  acid  it  does  not  give  the 
murexid  reaction. 

The  7-methyl  and  9 -methyl  purins  have  also  been  prepared.  Of 
especial  interest  is  the  fact  that  starting  out  with  methyl  uracil,  a 
pyrimidin  derivative,  Gabriel  and  Colman^  (1901),  succeeded  in 
converting  it  into  purin  bodies — 6-methyl  purin  and  6-2-methyl- 
amido  purin. 

The  pharmacological  action  of  purin  is  referred  to  on  p.  345. 

Adenin,  CgHgN^,  was  discovered  by  Kossel  in  1885.  It  was  pre- 
pared synthetically  by  Fischer^  (1)  by  treating  tri-chlor-purin  with 
ammonia  and  subsequent  reduction  of  the  dichlor-adeniu  ;  (2)  by 
similar  treatment  of  the  methyl  derivatives  of  oxy-di-chlor-purin. 
The  7-  and  9-methyl  adenins  were  incidentally  prepared. 

This  base  was  first  prepared  from  pancreatic  glands — hence  the 
term  adenin,  which  is  derived  from  the  Greek  word  adijv^  meaning 
a  gland.  It  has  since  been  shown  to  occur  together  with  guanin, 
hypoxanthin,  etc.,  as  a  decomposition  product  of  nuclein,  and,  there- 
fore, it  may  be  obtained  from  all  tissues  and  organs,  animal  or  vege- 
table, rich  in  nucleated  cells.  Accordingly  it  has  been  found  in  the 
kidneys,  spleen,  pancreatic,  thymus,  and  lymphatic  glands,  in  beer- 
yeast,  in  spermatic  fluids,  but  not  in  testicles  of  the  steer  ;  occurs 
also  in  tea-leaves.  In  the  latter  adenin  appears  to  exist  in  a  pre- 
formed condition,  since  it  can  be  extracted  without  the  use  of  acid 
reagents  (hypoxanthin  absent,  Kriiger,  1896).  Tea-extract  yields 
about  6  grams  of  adenin  per  liter  (Kriiger). 

The  thymus  gland,  as  a  prototype  of  embryonic,  highly  cellular 
tissue,  yields  a  considerable  amount  of  adenin,  but  no  xanthin 
(Inoko) ;  that  from  a  calf,  for  instance,  was  found  by  Schindler  to 
contain  0.18  per  cent.     The  thymus  nucleinic  acid  (adenylic  acid) 

1  Berichte,  34,  1246,  1256. 
^Berichte,  30,  2238. 


348  CHEMISTRY  OF  THE  LEUCOMAINS. 

was  at  first  believed  to  yield  only  adenin  but  eventually  was  found 
to  give  also  guanin  besides  cytosin  and  thymin  (Kossel  and  Neu- 
mann ^).  Although  the  pancreas  was  the  organ  from  which  origi- 
nally adenin  was  isolated,  yet  according  to  Bang  ^  it  yields  a  nucleinic 
acid  (guanylic)  which  on  decomposition  gives  rise  to  only  one  nuclein 
base — guanin,  besides  glycerin,  phosphoric  acid  and  pentose.  On  the 
other  hand  Levene  ^  obtained  from  a  pancreas  nucleinic  acid,  adenin, 
guanin  and  traces  of  xanthin  and  hypoxanthin.  He  also  found  that 
during  auto-digestion  of  the  pancreas  the  hexon  bases  and  uracil 
and  possibly  thymin  formed.  According  to  Kutscher  *  in  pancreatic 
and  yeast  auto-digestion  the  purin  bases  are  set  free  but  the  xanthin 
fraction  is  soon  destroyed,  leaving  only  adenin  and  guanin. 

From  tubercle  bacilli  Ruppel  isolated  a  protamin,  "  tuberculosa- 
min  "  and  a  nucleinic  acid,  tuberculinic  acid.  According  to  Levene 
the  nucleinic  acid  from  this  germ  is  less  stable  than  that  from  other 
sources.  It  contains  iron  and  on  decomposition  yields  guanin  and 
adenin. 

The  auto-digestion  of  yeast  is  essentially  the  same  as  that  of  the 
pancreas  and  like  the  latter  is  due  to  a  tryptic  ferment.  The  prod- 
ucts are  leucin,  tyrosin,  asparaginic  acid,  ammonia,  adenin,  guanin, 
at  times  hypoxanthin  and  xanthin,  also  the  three  hexon  bases. 
Carnin  and  butalanin  have  also  been  isolated.  Kutscher  has  also 
isolated  a  new  body,  CgHgN^O^ ,  which  probably  corresponds  to  the 
uracil  that  Levene  obtained  from  auto-digested  pancreas.  Geret  and 
Hahn^  had  previously  shown  that  the  yeast  plasma  contained  an 
energetic  proteolytic  enzyme  which  digested  fibrin,  egg  albumin  and 
pepton.  Leucin,  tyrosin  and  hypoxanthin  were  formed.  Similar 
enzymes  were  observed  in  tubercle  and  typhoid  bacilli,  in  sarcine, 
and  in  lupine  sprouts.  In  tryptic  digestion  of  nuclein  containing 
proteid  it  is  evident  that  adenin  and  other  xanthin  bases  may  be  set 
free  the  same  as  if  the  cleavage  had  been  brought  about  by  an  acid. 
The  bases  then  would  appear  in  the  "  antipepton  "  (p.  424).  In  auto- 
digested  adrenals  adenin  is  probably  present  (p.  386). 

Adenin,  guanin  and  xanthin  together  with  cholin,  diamins  (?), 
uric  acid  and  urea  have  been  obtained  from  brains  (Gulewitsch).^ 

From  10,000  liters  of  urine  Kriiger  and  Salomon  obtained  3.54  g. 
of  adenin  besides  large  amounts  of  xanthin  and  its  derivatives. 

It  has  also  been  observed  in  the  liver  and  urine  of  leucocythsemic 
patients  (Stadthagen) ;  its  occurrence  in  this  disease  will  be  readily 
understood  when  it  is  remembered  that  leukaemia  is  characterized  by 
the  presence  in  the  blood  of  an  unusual  proportion  of  the  nucleated 

^  Zeits.  physioI.  Chem.,  22,  74. 
^  Zeits.  physiol.  Chem.,  31,  411. 
^Zeits.  physiol.  Chem.,  32,  541. 
*Zeits.  physiol.  Chem.,  32,  66. 
^Berichte,  31,  2336  (1898). 
^  Zeits.  physiol.  Chem.,  27,  50. 


ADENIK  349 

white  blood  corpuscles,  which  owing  to  v^arious  unfavorable  condi- 
tions, become  destroyed  in  time,  and  the  contained  nuclein,  as  a  re- 
sult, splits  up  into  adenin  and  guanin.  These  two  bases  may,  there- 
fore, be  expected  in  all  pathological  conditions  where  there  is  an 
abnormal  accumulation  of  pus.  Indeed,  as  early  as  1865,  Naunyn 
extracted  from  pus,  obtained  from  the  pleural  cavity,  a  considerable 
quantity  of  a  substance  which  was  probably  either  adenin  or  guanin, 
or  both.  Neither  uric  acid  nor  xanthin  bases  in  recognizable 
amounts  are  present  in  fresh  human  blood  (100-300  c.c.) ;  both  are 
present  in  exudates  and  transudates  (Jaksch).  In  the  blood  of  leu- 
kaemics  and  in  blood  after  a  diet  of  thymus  glands  uric  acid  is  pres- 
ent in  increased  quantity  (Petreu). 

Adenin  does  not  occur,  or  only  in  minute  traces,  in  meat  extract ; 
and  in  this  it  resembles  guauin,  which  is  present  only  in  traces. 
This  may  be  due  to  the  fact  that  adenin  and  guanin  are  readily  con- 
verted into  hypoxanthin  and  xanthin  respectively,  as  has  been  shown 
in  the  putrefaction  experiments  of  Schindler.  This  conversion  of 
adenin  and  guanin  into  hypoxanthin  and  xanthin  takes  place  in  the 
pancreas  immediately  after  death,  so  that  the  amount  of  adenin  found 
may  be  quite  small.  They  may  be  considered  as  transitional  products 
of  cell  metabolism,  the  amido  group  contained  in  each  readily  being 
replaced  by  oxygen,  and  giving  rise  to  ammonia,  and  this  in  turn  to 
urea.  Kossel,  however,  explained  this  fact  on  the  ground  that  the 
muscle  tissue  is  very  poor  in  nucleated  cells,  i.  e.,  in  nuclein.  It 
would  seem  that  the  muscle  cell  in  losing  the  morphological  char- 
acter of  a  cell  has  also  suffered  a  corresponding  loss  in  its  chemical 
properties.  For  while  the  decomposition  products  of  nuclein — hy- 
poxanthin, xanthin,  phosphoric  acid,  etc. — are  found  in  the  muscle 
tissue,  they  do  not  exist  in  combination  as  they  do  in  the  nuclein 
molecules.  This  is  seen  in  the  fact  that  the  bases  exist  in  the  free 
condition,  since  they  can  be  extracted  by  water ;  and  again,  the  phos- 
phoric acid  is  present  in  the  muscle  tissue,  not  in  organic  combina- 
tion, but  as  a  salt.  In  the  nucleated  cell,  adenin,  guanin,  etc.,  do 
not  exist  in  a  free  condition,  but  form,  in  part  at  least,  with  albu- 
min and  phosphoric  acid,  a  loose  combination  which  is  readily  de- 
composed by  the  action  of  acids  at  the  boiling  temperature.  This 
same  change  takes  place  spontaneously  after  death.  It  is  quite  pos- 
sible that  the  existence  of  these  bases  in  muscles,  in  the  free  condi- 
tion, is  due  to  the  action  of  enzymes. 

There  can  be  no  doubt  that  adenin  and  guanin  play  an  important 
part  in  the  physiological  function  of  the  cell  nucleus,  which,  from 
recent  observations,  appears  to  be  necessary  to  the  formation  and 
building  up  of  organic  matter.  It  is  now  known  that  non-nucleated 
cells,  though  capable  of  living,  are  not  capable  of  reproduction.  We 
must  look,  therefore,  to  the  nucleus  as  the  seat  of  the  functional 
activity  of  the  cell — indeed,  of  the  entire  organism.     Nuclein,  the 


350  CHEMISTRY  OF  THE  LEUGOMAINS. 

parent-substance  of  adenin  and  guanin,  is  the  best  known  and  prob- 
ably most  important  constituent  of  the  nucleus,  and  as  such  it  has 
been  already  credited  with  a  direct  relation  to  the  reproductive 
powers  of  the  cell.  This  chemical  view  has  already  been  confirmed 
by  Zacharias,  who  showed  that  chromatin  of  histologists  is  identical 
with  nuclein.  More  recently  Mathews  ^  has  shown  that  the  chro- 
matin in  herring  spermatozoa  is  a  salt-like  combination  of  protamin 
and  nucleinic  acid.  Liebermann  has  questioned  nuclein  as  being 
the  source  of  xanthin  compounds,  but  in  this  he  is  not  supported  by 
the  mass  of  evidence.  In  the  case  of  birds,  however,  it  must  be 
conceded  that  uric  acid  results  by  the  oxidation  of  the  purin  bases 
and  above  all  by  synthesis  from  urea,  amido  acids,  etc.  In  mammals 
uric  acid  is  probably  wholly  derived  by  oxidation  of  the  purin  bases 
originating  from  nuclein.  Wiener,^  however,  has  rendered  it  probable 
that  the  synthetic  function  is  also  present  though  to  less  degree 
(p.  343).  Whether  purin  bases  as  well  as  uric  acid  may  be  formed 
synthetically  in  birds  remains  to  be  demonstrated. 

According  to  Minkowski  (1898)  adenin  is  a  powerful  poison. 
When  injected  subcutaneously  it  induces  increased  heart  action  and 
rapid  death.  On  feeding  it  affects  the  digestive  tract  and  the  kid- 
neys. The  constant  vomiting  induced  is  probably  due  to  the  intense 
inflammation  of  the  duodenal  mucous  membrane  which  may  lead  to 
actual  destruction  of  tissue.  The  effect  upon  the  kidney  is  seen  in 
the  presence  in  the  urine  of  casts,  albumin  and  of  minute  sphsero- 
liths  of  uric  acid.  On  section  the  kidney  shows  changes  similar  to 
those  observed  after  injection  of  urates.  The  cortex  especially 
reveals  the  presence  of  numerous  grayish  white  points  which  under 
the  microscope  are  found  to  consist  of  bundles  of  crystals  or  burs 
or  more  often  of  balls  which  show  a  concentric  or  radial  marking. 
These  deposits  are  undoubtedly  uric  acid  but  their  origin  is  un- 
known. The  excessive  uric  acid  may  be  ascribed  to  the  oxidation 
of  the  adenin  but  this  is  improbable.  It  is  more  likely  that  adenin 
favors  the  accumulation  of  uric  acid  by  interfering  with  the  de- 
struction of  this  product  in  the  liver.  The  studies  of  Wiener  have 
shown  that  the  crushed  liver  of  the  dog  (p.  343)  does  destroy  uric 
acid.  Adenin  is  not  as  poisonous  to  rabbits  and  man  as  to  dogs 
(Kriiger  and  Schmid,  1902). 

A  continued  diet  of  sweet-breads  which  as  is  known  are  rich  in 
antecedents  of  adenin  produced  symptoms  of  intoxications  such  as 
diarrhoea,  anorexia,  nausea,  headache,  malaise  and  abdominal  pains 
(Taylor).  The  observation  of  Kossel  that  adenin  is  in  part  excreted 
unchanged  has  been  confirmed  by  Minkowski  and  others. 

Lilienfeld,  in  his  study  of  the  chemistry  of  the  leucocytes,  showed 
that  the  nuclei  of  these  cells  contain  a  complex  body,  nucleohiston, 

'  Zeits.  physiol.  Chem. ,  23,  399. 

*  Archives  f.  ezp.  Path.  u.  Fharm.,  42,  898  (1899). 


ADENIK  351 

which  is  decomposed  by  acids  iuto  histon  and  leuconuclein.  The 
latter  in  turn  can  be  decomposed  into  albumin  and  nucleinic  acid, 
which  on  heating  with  mineral  acids  yields  phosphoric  acid,  and  the 
nuclein  bases  (adenin,  hypoxanthin)  and  unknown  products.  As 
Kossel  has  pointed  out,  it  is  probable  that  ordinary  nucleinic  acid 
is  a  mixture  of  several,  since  two  or  more  nuclein  bases  form  on 
decomposition.  The  existence,  however,  of  nucleohiston,  has  been 
questioned  by  Bang  and  others. 

Adenin,  when  crystallized  from  warm  or  impure  solutions,  is 
obtained  either  as  an  amorphous  substance,  pearly  plates,  or  in  the 
form  of  very  small  microscopic  needles  ;  from  dilute  cold  solutions  it 
separates  in  long,  needle-shaped  crystals  containing  three  molecules 
of  water.  This  water  of  crystallization  is  lost  on  exposure  to  the 
air  or  on  heating  to  53°,  and  the  crystals  become  opaque.  By  pre- 
cipitating a  concentrated  solution  of  the  hydrochlorid  with  ammonia 
adenin  may  be  obtained  as  anhydrous,  small  whetstone-shaped 
crystals,  which,  recrystallized  from  hot  water,  form  large,  regular, 
four-sided  pyramids,  single  or  bur-shaped.  It  is  soluble  in  about 
1086  parts  of  water  at  the  ordinary  temperature  ;  more  easily  in  hot 
water,  from  which  on  cooling  it  recrystallizes.  The  aqueous  solution 
possesses  a  neutral  reaction.  The  free  base  is  insoluble  in  ether, 
chloroform,  and  alcohol ;  soluble  in  glacial  acetic  acid,  and  some- 
what in  hot  alcohol.  It  dissolves  readily  in  mineral  acids,  yielding 
well  crystallizable  salts.  The  fixed  alkalis  dissolve  it  with  ease,  but 
on  neutralization  of  the  solution  it  is  reprecipitated  ;  from  such  solu- 
tions in  alkalis  anhydrous  large  crystals  are  thrown  down  by  acetic 
or  carbonic  acid  (Kriiger).  In  aqueous  ammonium  hydrate  it  is 
more  readily  soluble  than  guanin  (which  is  insoluble,  Schindler ; 
somewhat  soluble,  Wulff),  and  more  difficultly  soluble  than  hypo- 
xanthin— a  fact  which  is  made  use  of  to  effect  a  separation  from 
those  bases.     It  is  but  slightly  soluble  in  sodium  carbonate. 

Adenin  can  be  heated  to  278°  without  melting;  at  this  tempera- 
ture it  becomes  slightly  yellow,  and  yields  a  white  sublimate.  It 
can  be  completely  volatilized  without  decomposition,  by  heating  on 
an  oil-bath  at  220°  ;  the  sublimate  consists  of  pure,  white,  plumose 
needles  of  adenin,  but  at  250°  partial  decomposition  occurs,  and 
some  hydrocyanic  acid  forms.  According  to  Fischer,  when  heated 
rapidly,  in  a  capillary  in  a  paraffin-bath,  to  360°-365°  adenin  sud- 
denly melts  and  evolution  of  gas  takes  place.  When  heated  with 
potassium  hydrate  to  200°  on  an  oil-bath,  it  yields  a  considerable 
quantity  of  potassium  cyanid.  Adenin  is  quite  indifferent  to  the 
action  of  acids,  alkalis,  and  even  oxidizing  agents.  Thus,  it  may  be 
boiled  for  hours  with  baryta,  potash,  or  hydrochloric  acid,  without 
suffering  decomposition.  But  when  heated  with  dilute  hydrochloric 
acid  at  135°  for  several  days,  or  with  concentrated  hydrochloric 
acid,  in  a  sealed  tube  at  a  temperature  exceeding  100°,  adenin  is 


352  CHEMISTRY   OF  THE  LEUCOMAINS. 

completely    decomposed,    with    formation    of    carbonic    acid   and 
ammonia : 

C5H5N5  +  5H2O  +  50  =  5C0j  +  5NH3. 

When  heated  with  20  per  cent,  sulphuric  acid  in  an  autoclave  at 
150°  for  two  hours  adenin  is  decomposed  (Jones). 

On  heating  adenin  with  concentrated  hydrochloric  acid  to  180°— 
200°  for  12-14  hours,  Kriiger  obtained  ammonia,  carbon  dioxid, 
carbon  monoxid,  and  glycocoll.  The  carbon  monoxid  results  from 
the  splitting  up  of  formic  acid.  This  decomposition  is  strictly 
analogous  to  that  of  hypoxanthin,  xanthin,  etc. 

C5H5N5  +  8H,0  =  4NH3  +  CO,  +  2CH,02  +  CjHjNOa. 

The  free  base,  as  well  as  benzoyl  adenin,  is  unaffected  by  the  weak 
oxidizing  action  of  potassium  permanganate,  but  on  stronger  oxida- 
tion it  is  wholly  destroyed.  Bromin  water  produces  in  aqueous 
solutions  of  adenin  an  oily  precipitate  which,  on  contact  with  potas- 
sium hydrate  or  ammonia,  gives  a  beautiful  red  or  violet  color. 
Sodium  amalgam  and  zinc  chlorid  appear  to  have  no  action ;  but  on 
boiling  with  zinc  and  hydrochloric  acid  it  yields  a  very  unstable 
reduction  product  which,  in  the  presence  of  oxygen,  in  alkaline 
solution,  first  assumes  a  red  color  and  finally  throws  down  a  reddish- 
brown  precipitate.  This  brown  substance  appears  to  be  identical 
with  azulmic  acid,  which  has  been  known  for  a  long  time  as  a  product 
of  the  polymerization  of  hydrocyanic  acid. 

Adenin  and  hypoxanthin  do  not  give  the  xanthin  reaction ;  that  is 
to  say,  when  adenin  is  evaporated  on  a  water-bath  with  dilute  or 
fuming  nitric  acid  it  gives  a  white  residue  which  fails  to  give  any 
coloration  with  sodium,  ammonium,  or  barium  hydrate  (xanthin 
reaction).  Similarly,  it  does  not  give  the  so-called  Weidel's  reaction 
on  heating  with  fresh  chlorin  water  and  a  trace  of  nitric  acid  as  long 
as  gas  is  given  off,  then  evaporating  to  dryness  on  a  water-bath  and 
exposure  of  the  residue  to  an  ammoniacal  atmosphere.  In  this  re- 
spect it  resembles  hypoxanthin  which,  when  pure,  does  not  answer 
to  either  of  these  tests.  When  either  of  these  bases,  however,  is 
evaporated  on  a  water-bath  with  bromin  water  and  nitric  acid  a 
residue  is  obtained  which  with  alkalis  is  colored  red  (Kossel).  An- 
other test  for  adenin,  which  is  given  also  by  hypoxanthin,  but  not  by 
guanin,  caffein,  and  episarkin,  is  as  follows  :  The  substance  to  be 
tested  is  digested  for  half  an  hour  with  zinc  and  hydrochloric  acid 
in  a  test-tube  on  a  water-bath.  If  adenin  is  present,  the  solution 
will  assume  on  standing,  more  rapidly  on  shaking,  a  ruby-red  colora- 
tion which  in  time  disappears.  The  colorless  liquid  on  dilution  and 
addition  of  sodium  hydrate  becomes  again  red,  which  later  on  turns 
into  a  brownish-red  (Kossel).  This  reaction  depends  upon  the  for- 
mation of  a  reduction  product  which,  owing  to  its  unstable  nature, 


ADENIN.  353 

is  soon  oxidized  by  the  oxygen  of  the  atmosphere  into  a  brownish, 
amorphous  substance,  apparently  identical  with  aziilmic  acid. 

Ferric  chlorid  imparts  to  an  aqueous  solution  of  adenin  an  intense 
red  color  which  is  not  aifected  by  heating.  Copper  sulphate  pro- 
duces an  amorphous  grayish-blue  precipitate,  which  is  easily  soluble 
in  dilute  acids  and  ammonia.  The  light-blue  solution  in  fixed  alka- 
lis on  warming  gives  a  precipitate  of  copper  oxid. 

DrechseFs  reaction.  In  1892  Drechsel  showed  that  certain  xan- 
thin  bases  are  precipitated  by  an  ammoniacal  solution  of  cuprous 
chlorid ;  or  from  fixed  alkaline  solution  by  Fehling's  solution  in  the 
presence  of  a  reducing  substance.  In  addition  to  uric  acid,  which 
has  been  known  to  give  this  reaction,  xanthin,  guanin,  hypoxanthin, 
creatin,  and  creatinin,  the  latter  on  boiling,  reacted.  Balke  applied 
the  test  to  fixed  alkaline  solutions,  using  Fehling's  solution,  and  as 
reducing  substances  hydroxylamin  hydrochlorid,  or  dextrose.  He 
found  that  adenin,  hypoxanthin,  xanthin,  heteroxanthin,  paraxanthin, 
carnin,  protamin,  and  uric  acid  gave  precipitates,  whereas  theobro- 
min  and  caffein  did  not.  Kriiger  employed  copper  sulphate  and 
sodium  bisulphite,  the  advantage  being  that  the  precipitation  can 
take  place  in  neutral,  acid,  or  alkaline  solutions.  The  results  differ 
somewhat  with  the  kind  of  reducing  agent  employed.  Thus  copper 
sulphate  and  sodium  bisulphite  precipitate  uric  acid,  adenin,  methyl 
adenin,  hypoxanthin,  guanin,  also  dimethyl  hypoxanthin  from  cold 
concentrated  solution ;  theobromin,  caffein,  creatin,  creatinin  are 
not  precipitated.  With  copper  sulphate  and  sodium  hyposulphite, 
adenin,  methyl  adenin,  and  guanin  are  readily  precipitated  ;  hypo- 
xanthin only  on  heating  (separation  from  adenin),  whereas  the  other 
six  compounds  are  not  precipitated.  The  precipitates  are  soluble  in 
excess  of  sodium  hyposulphite. 

Adenin  and  hypoxanthin  can,  therefore,  be  completely  precipi- 
tated, especially  by  the  aid  of  heat,  from  their  solution  by  copper 
sulphate  and  sodium  bisulphite.  Hence  this  reagent  could  be  used 
as  a  substitute  for  ammoniacal  silver  solution  in  the  method  of  sepa- 
ration and  even  of  estimation  by  determining  either  the  amount  of 
copper,  or  of  nitrogen  by  Kjeldahl's  method.  The  adenin  precipi- 
tate is  colorless  and  gelatinous  ;  changes  on  exposure  to  a  light  or 
brownish  green,  and  on  drying  it  becomes  dark  green.  It  is  easily 
soluble  in  mineral  acids,  especially  nitric ;  slowly  soluble  in  hot 
acetic  acid.  It  is  not  decomposed  with  sodium  hydrate;  readily  de- 
composed with  alkali  sulphids,  and  is  readily  soluble  in  ammonia. 
It  is  soluble  in  about  200,000  parts  of  hot  water. 

On  treatment  with  nitrous  acid  it  is  converted  into  hypoxanthin 
according  to  the  equation  : 

C5H5N5  +  HNO,  =  C^H.N.O  +  Nj  +  H2O. 

Kossel  obtained  72  per  cent,  of  the  theoretical  yield.     Since  then 
23 


354  CHEMISTRY  OF  THE  LEUCOMAINS. 

Kriiger,  by  modifying  the  experimental  conditions,  adding  sodium 
nitrite  in  small  portions  to  a  solution  of  adenin  in  dilute  sulphuric 
acid  at  70°,  obtained  an  almost  quantitative  conversion. 

This  formation  of  hypoxanthin  from  adenin  is  analogous  to 
Strecker's  transformation  of  guanin  into  xanthin  by  a  similar  action 
of  nitrous  acid  (see  guanin).  In  both  cases  the  change  of  a  highly 
nitrogenized  into  a  less  nitrogenized  body  is  accomplished  by  replac- 
ing an  NHj  group  by  O.  The  change  is  somewhat  analogous  to  that 
seen  in  the  conversion  of  primary  amins  into  primary  alcohols.   Thus, 

CjHs.  NHj  +  HNO2  =  CjHjOH  +  N,  +  H,0. 
Ethylamik.         Ethyl  Alcohol. 

In  the  extraction  of  adenin  from  the  mother  liquor  of  tea  leaves 
after  removal  of  caffein,  if  urea  is  not  added  to  the  nitric  acid, 
nearly  one-half  of  the  adenin  may  be  converted  into  hypoxanthin. 
By  processes  of  putrefaction  adenin  is  converted  into  hypoxanthin 
and  guanin  into  xanthin  (Schindler).  A  similar  conversion  of 
adenin  and  guanin  takes  place  rapidly  in  the  pancreas  after  death 
and  it  is  not  unlikely  that  this  change  is  due  to  the  action  of  nitrous 
acid  produced  by  bacteria.  Adenin  undergoes  this  decomposition 
more  rapidly  than  do  the  other  purin  bases.  In  view  of  the  ease 
with  which  this  conversion  of  adenin  and  guanin  takes  place  it  is 
quite  probable  that  similar  changes  may  take  place  within  the  cell 
nucleus  proper  and  as  a  result  hypoxanthin  and  xanthin  form  in 
greater  or  less  amount.  The  formation  of  xanthin  from  guanin 
represents  the  conversion  of  a  guanidin  residue  into  a  urea  group. 
The  amido  group  in  all  probability  is  split  off  as  ammonia  and  goes 
to  make  urea. 

Adenin  unites  with  bases,  acids,  and  salts.  The  salts  of  adenin 
with  mineral  acids  can  be  recrystallized,  thus  differing  from  the  cor- 
responding salts  of  guanin  and  hypoxanthin,  which  are  dissociated 
by  the  action  of  water.  The  solutions  of  the  salts,  however,  show 
an  acid  reaction  to  litmus,  but  not  to  methyl  orange.  The  addition 
of  ammonia  to  concentrated  aqueous  solutions  of  its  salts  yields 
crystalline  adenin. 

The  hydrochlorid,  C5H5N5.HCI  +  2^2^)  forms  colorless,  trans- 
parent, strongly  refracting  crystals.  One  part  of  the  anhydrous 
salt  is  soluble  in  41.9  parts  of  cold  water.  Microscopically  it  is 
distinct  from  that  of  hypoxanthin  and  adenin-hypoxanthin.  From 
the  composition  of  the  gold  salt  it  is  highly  probable  that  a  hydro- 
chlorid, C5H5N5.2HCI,  exists  analogous  to  that  of  guanin.  In  the 
course  of  the  synthesis  of  adenin  Fischer  prepared  a  di-iodid. 

The  nitrate,  C5H5NJ..HNO3  -f  ^HjO,  crystallizes  from  the  aqueous 
solution  in  fine,  stellate  needles.  One  part  of  the  dry  salt  dissolves 
in  110.6  parts  of  water. 

The  sulphate,  (C,H,N,),.HjSO,  -|-  2Hp,  can  be  obtained  from  the 


ADENIN.  355 

aqueous  solution  in  two  diflPerent  crystalline  forms.  This  may  pos- 
sibly be  due  to  the  presence  of  the  adenin-hypoxanthin  compound 
(Bruhns).  It  is  easily  soluble  in  hot  water,  and  at  the  ordinary 
temperature  it  is  soluble  in  153  parts  of  water  (156,  Fischer). 

The  oxalate,  C^HgN^.CjHjO^  +  HjO,  is  obtained  by  dissolving 
adenin  in  hot,  dilute,  aqueous  oxalic  acid,  from  which  solution,  on 
cooling,  it  separates  as  a  voluminous  difficultly  soluble  precipitate  of 
roundish  masses  which  are  composed  of  long,  delicate  needles.  The 
oxalates  of  guanin,  hypoxanthin,  and  xanthin  are  more  easily  soluble 
than  that  of  adenin,  and  exhibit,  moreover,  a  different  appearance. 

Adenin  bichromate,  (C.H5N5)2.H2C207 .  This  compound  separates 
in  a  few  hours  from  a  mixture  of  adenin  and  chromic  acid  solutions 
in  well  formed  yellowish-red  crystals  (Bruhns).  According  to 
Kriiger,  it  forms  six-sided  plates,  is  easily  soluble  in  hot  water,  diffi- 
cultly in  cold,  and  is  unchanged  by  heating  to  150°.  The  corre- 
sponding salt  of  guanin  readily  dissociates. 

Adenin  metaphosphate,  C^H.N^.HPOg.  According  to  Kossel, 
adenin  is  not  precipitated  with  metaphosphoric  acid,  but  this  is  not 
strictly  true.  Aqueous,  or  even  cold  saturated  solutions  of  adenin  give 
on  the  addition  of  a  few  drops  of  metaphosphoric  acid  an  amorphous 
precipitate,  appearing  under  the  microscope  as  fine  round  granules  or 
extremely  thin  membranous  masses.  It  has  not  been  obtained  in  a 
crystalline  condition.  Like  the  corresponding  guanin  compound,  it  is 
difficultly  soluble  in  cold  water.  It  is  easily  soluble  in  alkalis  and  in 
ammonia  ;  is  more  or  less  soluble  in  dilute  acids  according  to  the  con- 
centration, and  is  soluble  in  excess  of  metaphosphoric  acid.  Hence 
a  strongly  acid,  not  too  concentrated,  solution  of  adenin  is  not  pre- 
cipitated (Wulff).  Adenin  is  precipitated  less  completely  than 
guanin,  whereas  hypoxanthin  does  not  give  a  difficultly  soluble 
metaphosphate. 

The  chloracetate,  CjHjNg.ClCHj.COjH,  was  prepared  by  Kriiger 
by  adding  an  excess  of  chloracetic  acid  to  a  hot  aqueous  solution  of 
adenin.  On  cooling  it  crystallizes  in  right-angled  plates  and  in  stel- 
late four-sided  prisms.  It  is  easily  soluble  in  water  and  in  hot 
aqueous  alcohol;  difficultly  in  cold  alcohol.  At  162°-163°  it 
melts,  giving  off  hydrochloric  acid  and  forming  a  yellowish-red  fluid 
which  gradually  becomes  intensely  red. 

Potassium  ferro-  and  ferricyanid  produce  no  precipitate  in  a  solu- 
tion of  adenin,  but  if  acetic  acid  is  then  added  the  former  gives 
rise  to  a  precipitate  of  thin  plates ;  the  latter,  to  a  precipitate  of 
light-brown  crystals  grouped  in  bunches  (Kriiger).  According  to 
Bruhns,  adenin  gives  with  potassium  ferricyanid  brownish-green 
needles. 

The  picrate,  C5lI,N5.CgH2(N02)30H  +  Hp,  is  thrown  down  as  a 
bright-yellow  flocculent  precipitate  when  aqueous  solutions  of  adenin 
salts  are  treated  with  sodium  picrate.     Recrystallized  from  hot  water 


356  CHEMISTRY  OF  THE  LEUCOMAINS. 

it  forms  bright-yellow,  very  voluminous  bunches  of  long,  fine  needles 
which,  on  drying,  acquire  a  silky  luster  and  form  a  felted  mass.  It 
is  difficultly  soluble  in  cold  water  (1:3500);  more  readily  in  hot 
water  and  in  alcohol  (96  per  cent.);  is  insoluble  in  dilute  acids.  It 
dissolves  readily  in  a  solution  of  sodium  phosphate,  from  which  solu- 
tion it  is  precipitated  by  hydrochloric  acid.  Other  salts  of  adenin, 
as  the  metaphosphate,  behave  in  the  same  way.  Uric  acid  is  also 
dissolved  by  sodium  phosphate  (Wulflf).  The  water  of  crystalliza- 
tion is  not  lost  on  exposure  to  air,  but  is  driven  off  at  100°;  the  salt 
then  remains  unchanged  even  at  220°.  A  cold  concentrated  aqueous 
solution  of  the  salt  treated  with  one-tenth  its  volume  of  cold  concen- 
trated solution  of  sodium  picrate  produces  a  precipitate  of  short,  fine 
needles,  consisting  of  most  of  the  adenin  picrate  (five-sevenths). 
The  solubility  of  the  picrate  can  thus  be  reduced  to  as  low  as 
1:13750,  and  on  this  fact  is  based  the  quantitative  method  of 
Bruhns.  The  salt  can  also  be  obtained  in  its  characteristic  groups 
by  combining  cold  saturated  aqueous  adenin  solution  (1 :  1086)  with 
picric  acid ;  with  sodium  picrate,  however,  adenin  gives  no  precipi- 
tate, since  the  picrate  is  soluble  in  an  equivalent  quantity  of  sodium 
hydrate.  Thus  is  explained  Kossel's  statement  that  adenin  forms 
an  easily  soluble  compound  with  picric  acid.  Heated  on  platinum 
foil  it  burns  slowly  and  leaves  considerable  carbon  residue.  The 
very  bright  yellow  color  of  the  salt  serves  to  distinguish  it  from  most 
of  the  other  picrates,  especially  guanin  picrate.  Adenin  may  be  iso- 
lated from  its  picrate  by  extraction  of  the  hydrochloric  acid  solution 
with  ether,  by  precipitation  of  the  ammoniacal  solution  with  silver 
nitrate,  and  best,  according  to  Kriiger,  by  dissolving  the  picrate  in 
hot  dilute  ammonia,  and  when  cold  precipitating  most  of  the  picric 
acid  with  ammoniacal  copper  sulphate  solution.  The  filtrate  can 
then  be  evaporated,  dissolved  in  dilute  HgSO^,  and  the  last  traces  of 
picric  acid  removed  with  ether. 

It  may  be  noted  that  adenin  and  guanin  form  difficultly  soluble 
picrates,  whereas  xanthin  and  hypoxanthin  form  relatively  easily 
soluble  compounds. 

The  platinochlorid,  (C5H5N5.HCl)2PtCl^,  crystaUizes  from  dilute 
aqueous  solution  in  small  yellow  needles.  The  concentrated  aqueous 
solution  of  this  salt,  when  boiled  for  some  time,  decomposes,  Avith 
the  separation  of  a  clear,  yellow  powder,  which  is  but  slightly  soluble 
in  cold  water,  and  has  the  composition  Cj-HgNg.HCl.PtCl^ . 

The  aurochlorid,  on  evaporation,  yields  very  characteristic  forms. 
It  has  been  more  recently  studied  by  Wultf,  and  found  to  possess 
the  formula  aH5N,.(HCl)2.AuCl3  -f  H^O.  From  the  hydrochloric 
acid  solution  of  adenin  and  gold  chlorid,  on  sufficient  concentration, 
or  from  dilute  solutions  by  gradual  evaporation,  it  separates  in 
bright,  well  formed  orange-colored  crystals,  which  may  attain  a 
length  of  1.2  cm.      As  pointed  out    by  Kossel,  this    salt   is    well 


ADENIN.  367 

adapted  for  the  qualitative  recognition  of  adenin,  especially  in  the 
presence  of  guanin,  which  gives  no  such  compound. 

Adenin  lead  was  prepared  by  Kriiger  by  adding  a  solution  of 
adenin  and  sodium  hydrate  to  an  aqueous  solution  of  lead  acetate. 
It  forms  lusterless  needle-shaped  crystals.  The  composition  appears 
to  be  C^HgPbNj .  On  friction  it  becomes  strongly  electric.  Heated 
with  methyl  iodid  it  gives  rise  to  addition  products  (see  page  363). 

The  silver  salt  of  adenin,  C^H^AgNj,  is  formed  when  silver 
nitrate  is  added  in  molecular  proportion  to  a  boiling  ammoniacal 
solution  of  adenin.  On  heating  this  compound  for  thirteen  hours 
at  130°  with  methyl  iodid  no  appreciable  change  results  (Kriiger), 
although  Thoiss  obtained  a  compound,  presumably  a  methyl  addition 
product.  An  excess  of  silver  nitrate  produces,  in  the  cold,  the  com- 
pound C5H3Ag2jS'5  +  HjO,  which  is  converted  slowly  in  the  cold, 
immediately  on  warming,  into  the  other  salt,  according  to  the 
equation  : 

2(C,H3Ag,N,  +  H3O)  =  2C,H,AgN,  +  Agp  +  H.O. 

Owing  to  this  instability  the  two  compounds  are  always  found  to- 
gether in  varying  proportion.  Both  are  difficultly  soluble  in  water, 
and  in  ammonia  even  at  the  boiling-point.  The  precipitation  of 
adenin  by  an  ammoniacal  silver  solution  is  complete,  and  is  there- 
fore available  for  quantitative  estimation.  The  precipitate  of  adenin, 
as  well  as  of  other  xanthin  bases,  is  soluble  in  excess  of  sodium 
hyposulphite  (Kossel). 

Adenin  silver  nitrate,  C^HjN^.AgNOg  (Ag=  35.4  percent.),  cor- 
responds to  the  similar  hypoxanthin  and  guanin  salts.  It  is  obtained 
by  dissolving  the  above  silver  compound  in  hot  nitric  acid ;  and 
from  this  solution,  on  cooling,  it  separates  in  needle-shaped  crystals, 
which  are  not  permanent.  This  lack  of  stability,  as  compared  with 
the  permanent  hypoxanthin  silver  nitrate,  was  first  pointed  out  by 
Kossel  and  was  thought  to  be  due  to  the  loss  of  nitric  acid  in  wash- 
ing, and  also  by  heating  at  100°.  Bruhns,  however,  has  shown  that 
the  acidity  of  the  wash-water  is  indicated  by  litmus,  but  not  by 
methyl  orange,  which  is  not  colored  red  by  silver  nitrate.  It  would 
seem  that  adenin,  as  well  as  hypoxanthin,  and  possibly  xanthin, 
form  silver  compounds  containing  one  and  two  molecules  of  silver 
nitrate ;  the  greater  the  quantity  of  silver  nitrate  used  the  higher  is 
the  per  cent,  of  silver,  i.  e.,  the  more  of  the  latter  compound  is 
formed.  These  are  very  unstable,  and  are  decomposed  by  dilute 
nitric  acid,  more  so  by  water,  into  silver  nitrate,  and  the  com- 
pound containing  one  molecule  of  silver  nitrate.  We  have  in  this 
behavior  an  interesting  case  of  mass  action  and  chemical  equili- 
brium between  adenin,  silver  nitrate,  nitric  acid  and  water.  Ammo- 
nium hydrate  removes  the  nitric  acid  from  this  as  easily  as  from  the 
hypoxanthin  compound,  and  there  is  formed,  according  to  the  com- 


358  CHEMISTRY  OF  THE  LEUCOMAINS. 

position  of  the  original  salt,  a  varying  mixture  of  CgH^AgNg  and 
CgHgAgjNg  +  HgO.  The  solubility  in  nitric  acid  is  about  the  same 
as  that  of  hypoxanthin  silver  nitrate. 

Adenin  silver  picrate,  C5H^AgN5.CgH2(N02)30H  +  Hp,  is  ob- 
tained as  an  amorphous  voluminous  yellow  precipitate  when  silver 
nitrate  is  added  to  a  cold  aqueous  solution  of  adenin  picrate.  If 
the  latter  solution  is  previously  raised  to  the  boiling-point,  the  pre- 
cipitate soon  becomes  crystalline  and  rapidly  subsides.  The  adenin 
can  thus  be  almost  wholly  removed  from  solution.  The  crystalline 
form  loses  its  water  of  crystallization  at  120°,  while  the  amorphous 
form  does  not  appreciably  decrease  in  weight,  and  its  composition 
does  not  appear  to  be  so  constant  as  that  of  the  corresponding  hypo- 
xanthin compound.  On  treatment  with  ammonium  hydrate  the 
picric  acid  is  removed,  and  adenin  silver,  CgH^AgNg ,  is  left,  stained 
yellow  by  picric  acid. 

Adenin  mercury  picrate,  (C5H^Ng)2Hg.2CgH2(N020)30H,  can  be 
prepared  by  treating  a  hot  concentrated  aqueous  solution  of  adenin 
picrate  with  an  excess  of  sodium  picrate,  and  then  with  mercuric 
chlorid.  It  forms  a  yellow  granular,  crystalline  precipitate  (micro- 
scopic needles)  which  rapidly  subsides  and  increases  in  quantity  as 
the  solution  cools.  Its  composition  apparently  varies,  containing  one 
to  two  molecules  of  water,  according  to  the  temperature  of  the  solu- 
tion. One  molecule  is  given  off  at  100°  and  the  second  at  105°— 120°. 
The  latter  preparation,  then,  on  exposure  to  the  air,  rapidly  absorbs 
one  molecule  of  water.  The  object  of  the  sodium  picrate  in  the 
precipitation  is  to  combine  with  the  hydrochloric  acid,  which  is  set 
free.  The  precipitate  produced  by  mercuric  chlorid  in  cold  adenin 
picrate  solution  shows  yellow  and  white  granules,  and  is  not  homo- 
geneous. Bruhns  considers  it  to  be  a  mixture  of  the  adenin  mer- 
cury picrate  and  the  compound  CgH^NgllggClj ;  if  sodium  picrate  is 
added,  however,  the  pure  adenin  mercury  picrate  forms,  since  no 
hydrochloric  acid  is  set  free. 

Adenin  mercuric  chlorid,  C^H^NgllgCl,  is  thrown  down  as  a  white, 
finely  granular  precipitate  when  a  boiling  aqueous  adenin  solution 
is  treated  gradually  with  concentrated  mercuric  chlorid  solution 
(Bruhns).  On  neutralizing  the  filtrate  from  this  precipitate  a  second 
deposit  forms.  According  to  Kriiger,  the  reaction  that  takes  place 
is  as  follows  : 

2C,H,N,  +  2HgCl2  =  C,H,N,.HgCl  +  C,H5N,.HCl.HgCl2. 

C5H5N5.HCl.HgCl2+ Na2C03=  C5H,N,.HgCl  +  2NaCl  +  CO^  +  Hp. 

Heated  with  alkyl  iodids  it  does  not  give  rise  to  substitution  com- 
pounds. Free  hydrochloric  acid  is  indicated  by  the  reaction  with 
methyl  orange.  Treated  with  ammonium  hydrate  the  chlorin  is  re- 
moved, and  there  is  formed  apparently  the  compound  CgH^Nj-HgOH. 
If  dissolved  in  warm  dilute  hydrochloric  acid  and  allowed  to  crystal- 


ADENIN.  359 

lize,  the  double  salt  CjHgNj.HCl.HgClg  +  SHp  separates  in  long, 
stellate,  silky  needles. 

Another  mercury  compound,  C5H^N5Hg2Cl3 ,  is  obtained  when  the 
precipitation  takes  place  in  the  cold.  The  precipitate  is  white, 
flocculeut,  and  anhydrous.  In  this  reaction,  as  above,  for  each 
adenin  molecule  an  equivalent  of  hydrochloric  acid  is  set  free. 
This  same  body  is  also  produced  when  an  adenin  solution  is  boiled 
with  a  large  excess  of  mercuric  chlorid  and  as  little  hydrochloric 
acid  as  possible  to  effect  solution.  On  cooling  small  stellate  needles 
separate  out,  which  do  not  lose  their  weight  at  110°.  It  can  also 
be  obtained  by  boiling  the  following  compounds  with  water. 

When  adenin  is  boiled  with  a  large  excess  of  mercuric  chlorid  and 
much  hydrochloric  acid  to  dissolve  completely  the  precipitate  that 
first  forms,  there  is  deposited  on  cooling  a  crystalline  product,  which 
is  variable  in  its  composition,  and  apparently  consists  of  double  salts 
of  adenin  and  mercuric  chlorid,  such  as  CgH.Ng.HCl.SHgCIg  and 
CgHgNg.HCl.GHgCl^.  On  boiling  with  water  these  rapidly  decom- 
pose, forming  the  compound  C5H^N5.IIg2Cl3 .  The  formation  of  a 
double  salt,  qHgN^.HCl.Hg^Cl^  +  2H2O,  is  described  above. 

Adenin  mercury  cyanid,  (C.H5N5)2.Hg(CN)2,  separates  as  stellate 
needles  and  plates  when  a  mixture  of  hot  solutions  of  adenin  and 
mercuric  cyanid  are  allowed  to  cool. 

An  adenin  bismuth  iodid,  C5H5Ng.HI.2Bil3  +  211^0,  is  obtained 
when  an  aqueous  adenin  solution  is  treated  with  potassium  bismuth 
iodid  containing  free  hydriodic  acid.  The  heavy  precipitate,  which 
in  color  resembles  carbon  monoxid  haemoglobin,  consists  of  micro- 
scopic glittering  red  needles.  On  contact  with  much  water  it 
partly  decomposes,  forming  light  reddish-yellow  amorphous  floccules, 
which  become  darkish  brown  at  100°. 

Chlor-adenin  has  not  been  obtained,  since  chlorin  passed  over 
dry  adenin  in  the  cold,  or  at  100°,  or  into  a  boiling  chloroformic 
suspension  of  adenin  is  without  effect.  Phosphorus  pentachlorid 
heated  with  adenin  at  160°- 170°  for  some  hours  gave  a  light-brown 
body  of  uncertain  composition. 

The  synthetic  di-chlor  adenin  is  derived  from  tri-chlor  purin. 

Brom-adenin.  By  treating  well  dried  adenin  with  excess  of  dried 
bromin  a  dark-red  body  is  obtained  which  appears  to  contain  six 
atoms  of  bromin,  C^HgNg.Brg  (Bruhns).  On  mere  exposure  to  the 
air,  more  rapidly  on  heating  at  100°- 120°,  it  becomes  light-yellow 
and  decomposes,  yielding  bromin,  brom-adenin,  CgH^BrNg,  and  its 
hydrobromid,  Cgll^BrNg.HBr.  Brom-adenin  is  white,  difficultly 
soluble  in  cold  water  (1  :  10,000),  more  readily  in  hot  water,  very 
easily  in  ammonia  and  in  fixed  alkalis.  It  crystallizes  from  water 
or  dilute  ammonia  in  stellate  needles  or  very  thin  plates  which, 
when  dried  in  air,  often  assume  a  silky  luster.  The  crystals  con- 
tain a  variable  amount  of  water  depending  on  the  temperature  at 


360  CHEMISTRY  OF  THE  LEUCOMAINS. 

which  the  crystallization  takes  place.  Thus,  the  crystals  may  con- 
tain almost  two  molecules  of  water,  whereas  when  crystallization 
occurs  at  above  60°  the  crystals  are  anhydrous.  It  is  a  rather 
strong  base  and  forms  well  characterized  salts  which  are  difficultly 
soluble  in  cold  water,  more  easily  in  the  presence  of  an  excess  of 
acid,  from  which  it  is  thrown  down  as  a  white  micro-crystalline  pre- 
cipitate by  addition  of  ammonia.  It  is  also  formed  from  the  orig- 
inal dark-red  body  by  treatment  with  sodium  bisulphite,  or  better 
by  dissolving  the  body  in  ammonium  hydrate,  or,  according  to 
Kriiger,  by  heating  to  130°,  then  dissolving  in  sodium  hydrate  and 
precipitating  with  carbonic  or  acetic  acid.  It  is  only  difficultly  at- 
tacked by  boiling  alcohol  or  aqueous  potash  or  alcoholic  ammonia. 
The  atom  of  bromin  cannot  therefore  be  replaced  by  an  amido  or 
by  a  hydroxyl  group.  Sodium  alcoholate  heated  with  brom-adenin 
at  145°  for  hours  has  no  effect. 

Brom-adenin  is  very  easily  and  completely  changed  to  adenin  by 
the  action  of  sodium  amalgam  in  the  cold,  or  by  boiling  for  several 
hours  with  zinc  dust.  No  azulmic  acid  is  formed.  It  is  not  affected 
by  iron  dust  (Bruhns).  According  to  Kriiger,  it  is  affected  by  heat- 
ing with  concentrated  potash  at  180°- 190°,  and  the  bromin  is  not 
replaceable  by  radicals  as  phenol. 

The  study  by  Bruhns  of  the  decomposition  of  the  dark-red  body, 
mentioned  above,  has  shown  that  it  is  very  probably  a  hydrobromid 
of  brom-adenin,  tetra-bromid,  CgH^BrNg.Br^.HBr.  According  to 
Kriiger,  this  compound  does  not  always  form  by  the  addition  of 
bromin  to  adenin.  Ordinarily  the  hydrobromid  of  brom-adenin 
forms,  unless  a  very  large  excess  of  bromin  is  used.  Compounds 
similar  to  brom-adenin  are  formed  by  hypoxanthin,  guanin,  xanthin, 
and  caffein.  Azulmic  acid  reacts  with  bromin  in  much  the  same 
way  as  adenin. 

The  hydrochlorid,  sulphate,  and  nitrate  of  brom-adenin  have 
been  prepared  and  analyzed  by  Bruhns. 

Brom-adenin  picrate,  C5lI^BrN5.CgH2(N02)30H  -|-  Hp,  resembles 
that  of  adenin,  but  is  more  voluminous.  It  is  precipitated  under 
the  same  conditions  as  adenin.  The  solubility  in  cold  water  is 
about  the  same  (1:3220).  It  is  likewise  almost  completely  thrown 
out  of  solution  by  sodium  picrate.  Under  the  microscope,  how- 
ever, it  can  be  readily  distinguished  from  adenin  picrate,  since  it 
does  not  form  distinct  crystals,  but  rather  bundles  of  thin  thread-like 
needles. 

The  metal  derivatives  of  brom-adenin  are  analogous  to  those  of 
adenin.  Thus,  ammoniacal  silver  solution  gives  rise  to  a  mixture 
of  CjHgAgBrNj  and  C,H2Ag2BrN5.H20.  Silver  nitrate  produces  a 
gelatinous  precipitate  which,  like  the  adenin  silver  nitrate,  has  an  in- 
constant composition  ;  on  careful  heating  with  nitric  acid  (1.1  sp.  g.) 
it  can  be  obtained  in  needles  which  resemble  exactly  those  of  the 


ADENIN.  361 

adenin  compound.  Prolonged  boiling  with  nitric  acid  results  in  the 
separation  of  silver  bromid.  Mercuric  chlorid,  cadmium  chlorid, 
potassium  bismuth  iodid,  etc.,  give  precipitates  with  brom-adenin 
the  same  as  with  adenin. 

Brom-adenin  gives  the  xanthin  reaction,  whereas  adenin  itself 
does  not.  Thus,  if  evaporated  with  strong  nitric  acid  on  the  water- 
bath  to  dryness,  and  the  cold  yellowish  or  reddish  residue  is  touched 
with  sodium  hydrate,  a  bluish-violet  color  forms.  With  ammonia  it 
is  a  purple-red  ;   with  baryta  water  a  pure  violet. 

Dry  chlorin  gas  passed  over  warm  dry  brom-adenin  has  no  effect. 
If,  however,  the  brom-adenin  is  moist,  decomposition  and  solution 
result.  On  evaporation  of  the  solution  the  residue  gives  with  potas- 
sium hydrate  an  intense  violet-red  color ;  baryta  produces  a  bluish- 
green  precipitate. 

It  is  therefore  evident  from  the  above  reactions  with  nitric  acid 
and  chlorin  that  brom-adenin  is  more  readily  destroyed  or  oxidized 
than  adenin.  Inasmuch  as  all  attempts  at  obtaining  oxidation 
products  of  adenin  which  would  shed  light  on  its  constitution  failed, 
the  study  of  the  oxidation  products  of  brom-adenin  therefore  pos- 
sessed special  interest.  Kriiger  succeeded  in  oxidizing  brom-adenin 
with  hydrochloric  acid  and  potassium  chlorate  in  warm  solution, 
into  alloxan,  urea,  and  oxalic  acid.  A  reddish  substance  which 
dissolved  in  alkalis  with  a  purple-red  color  was  also  produced  in 
small  amounts.  Its  alkaline  solution  gave  a  dirty-blue  precipitate 
with  baryta.  The  amount  of  alloxan  found  was  very  small ;  indeed, 
in  one  experiment  it  was  entirely  absent.  Nevertheless,  it  was 
sufficient  to  prove  that  in  adenin,  and  hence  in  hypoxanthin,  an 
alloxan  group  and  probably  an  urea  residue  were  present  as  in  uric 
acid  and  xanthin. 

When  adenin  is  treated  with  zinc  and  hydrochloric  acid  in  the 
cold  it  forms  a  difficultly  soluble  crystalline  double  salt  which  has 
not  been  obtained  in  the  pure  state.  This  double  salt  is  not  obtained 
by  direct  treatment  of  adenin  hydrochlorid  with  zinc  chlorid. 

One  of  the  hydrogen  atoms  of  adenin  is  capable  of  replacement 
by  organic  radicals,  as  seen  from  the  following  compounds  : 

Acetyl  adenin,  C.H^Nj.CO.CH,,  can  be  obtained  by  heating  the 
anhydrous  base  with  an  excess  of  acetic  anhydrid  for  some  time,  in 
an  oil-bath,  at  130°.  It  crystallizes  in  small  white  scales  which 
dissolve  but  slightly  in  cold  water  and  in  alcohol ;  more  readily  in 
hot  water,  in  dilute  acids  and  alkalis.  Heated  to  260°,  it  becomes 
yellow,  but  does  not  melt. 

Benzoyl  adenin,  Cgll^Ng.CO.CgHg,  is  obtained  by  the  action  of 
benzoic  anhydrid,  but  not  of  benzoyl  chlorid  or  adenin.  It  crystal- 
lizes from  water  in  long,  lustrous,  thin  needles  which  sometimes  are 
grouped  in  bundles  and  melt  at  234°- 235°.  It  is  easily  soluble  in 
hot  alcohol,  from  which  it  recrystallizes  on  cooling ;  also  in  dilute 


362  CHEMISTRY  OF  THE  LEUCOMAINS. 

acids  and  in  ammonia.  With  ammoniacal  silver  nitrate  it  gives  a 
precipitate  resembling  that  of  adenin,  but  is  more  readily  soluble  in 
ammonia.  This  compound  is  quite  stable,  since  it  decomposes  very 
slowly  on  boiling  with  hydrochloric  acid  ;  on  protracted  boiling  with 
water  it  is  changed  into  adenin  and  benzoic  acid. 

Mono-benzyl  adenin,  C.H^N5.CH2.CgH5,  was  obtained  by  Thoiss 
by  heating  well  dried  adenin  with  benzyl  chlorid  to  boiling  (178°) 
on  an  oil-bath.  It  can  also  be  obtained,  according  to  Kriiger,  by 
heating  adenin  in  a  flask  with  benzyl  chlorid  in  a  sulphuric  acid 
bath  ;  also  by  heating  adenin  with  alcohol,  potassium  hydrate,  and 
benzyl  chlorid  under  an  inverted  condenser.  It  crystallizes  from 
alcohol  in  short,  glistening  prisms,  frequently  in  small  pointed  crys- 
tals grouped  in  plate-like  aggregations.  A  ten  per  cent,  alcoholic 
solution  gives  reactions  with  silver  nitrate,  ammoniacal  silver  solu- 
tion, mercuric  chlorid,  picric  acid  and  platinum  chlorid.  Gold 
chlorid  gives  no  precipitate.  The  compound  forms  pure  white  micro- 
scopic crystals  and  melts  at  259°.  It  is  easily  soluble  in  hot 
water  and  in  hot  alcohol ;  but  little  in  ether.  Its  solubility  in  water 
at  15°  is  1:  2250  ;  in  water  at  100°  is  1:  320.  With  acids  it  forms 
salts  from  which  alkalis  throw  down  the  base.  The  hydrochlorid 
0^11^(0^11^)^5.1101,  forms  fine  glassy  needles  or  four-sided  glassy 
prisms  with  inclined  end-surfaces,  which  are  readily  soluble  in  alco- 
hol and  in  water,  but  not  in  ether.  The  sulphate  and  nitrate  pos- 
sess similar  properties.  The  sulphate,  {C^IQ^H^)^ ^^  .HgSO^ ,  forms 
glassy  long  prisms  containing  five  molecules  of  water,  four  of  which 
easily  pass  ofFat  100°,  and  the  fifth  at  1 10°.  Like  adenin,  it  yields  a 
silver  compound  which  is  insoluble  in  ammonia.  On  reduction  with 
zinc  and  hydrochloric  acid  it  forms  an  amorphous  red  unstable  com- 
pound. Treated  with  nitrous  acid,  benzyl  adenin  is  reduced  to 
benzyl  hypoxanthin,  thus  showing  that  the  benzyl  group  replaces  a 
hydrogen  atom  in  the  group  Ogll^N^,  which  Kossel  has  called 
adenyl  (see  page  336). 

Benzyl  adenin  picrate,  0j2HjjN5.0gIl2(]S'O2)3OH,  is  obtained  as 
fine  felted  yellow  needles,  which  are  fairly  soluble  in  water  and  in 
alcohol ;  insoluble  in  ether. 

Like  adenin,  the  benzyl  compound  is  very  resistant  to  oxidation 
with  potassium  permanganate.  On  treatment  with  sulphuric  acid 
and  chromic  acid  a  part  is  completely  oxidized  and  the  remainder  is 
unchanged.  Bromin  acts  energetically,  forming  a  dark-red  sticky 
mass,  which  at  120°  only  gradually  gives  off  a  part  of  the  bromin 
and  becomes  dark-yellow  in  color  and  firmer  in  consistency.  Appa- 
rently four  atoms  of  bromin  unite  with  one  molecule. 

On  decomposition  with  concentrated  hydrochloric  acid  at  180°- 
200°  it  yields  glycocoll,  volatile  bases,  and  a  resinous  body,  Oj^Hj^, 
identical  with  that  obtained  by  Oannizzaro  by  the  action  of  dehy- 
drating agents  on  benzyl  alcohol  (Kriiger). 


ADENIN.  368 

Dibenzyl  adenin,  C5H3(C7H^)2Ng ,  is  produced,  according  to  Kriiger, 
in  small  amount  in  the  preparation  of  the  mono-benzyl  compound.  It 
is  best  obtained  by  treating  mono-benzyl  adenin  with  benzyl  chlorid ; 
or  by  the  action  of  benzyl  chlorid  on  an  alcoholic  solution  of  adenin 
and  potassium  hydrate.  The  free  base  is  obtained  by  precipitating 
a  solution  of  the  hydrochlorid  with  ammonium  hydrate.  It  forms 
fine  silky  needles  which  melt  at  171°  to  a  yellow  fluid.  It  is  easily 
soluble  in  ether,  very  easily  in  alcohol.  In  cold  water  at  13.5°  the 
solubility  is  1 :  13,300 ;  in  water  at  100°  it  is  1 :  1300.  A  1  per  cent, 
alcoholic  solution  gives  reactions  with  silver  nitrate,  ammoniacal 
silver  solution,  mercuric  chlorid,  copper  sulphate,  platinum  chlorid, 
and  picric  acid.  Gold  chlorid,  lead  acetate,  basic  lead  acetate,  give 
no  precipitate. 

The  hydrochlorid,  C5H3(C^H^)2N,.HC1,  crystallizes  in  fine  silky 
needles,  frequently  in  long  prisms  with  a  silky  luster  resembling  that 
of  caffein.  It  is  easily  soluble  in  water  and  in  alcohol ;  insoluble  in 
ether.  It  is  thrown  out  of  water  solution,  in  part,  by  the  addition 
of  hydrochloric  acid — a  reaction  not  given  by  the  mono-benzyl  com- 
pound.    The  melting-point  is  at  219°- 220°. 

The  nitrate,  05113(07117 )2N5  .HNO3 ,  crystallizes  in  fine,  long  glis- 
tening needles,  which  are  difficultly  soluble  in  cold  dilute  nitric  acid. 
It  melts  at  167°  with  evolution  of  gas.  On  decomposition  with  con- 
centrated hydrochloric  acid  at  180°- 200°  it  yields  the  same  prod- 
ucts as  mono-benzyl  adenin  (Kriiger). 

9-methyl  adenin,  05H^(OH3)N5 ,  was  first  prepared  in  a  condition 
of  purity  by  Kriiger.  In  1897,  Fischer'  prepared  the  7-  and  9- 
methyl  adenins  synthetically.  The  methyl  adenin  described  by 
Thoiss  and  prepared  by  the  action  of  methyl  iodid  on  adenin-silver 
at  100°  is  probably  an  addition  product,  not  methyl  adenin.  The 
introduction  of  the  methyl  group  in  this  way  does  not  take  place 
readily,  according  to  Kriiger,  owing  to  the  formation  of  addition 
products.  This  addition  of  methyl  iodid  can  be  overcome  readily 
by  the  presence  of  sodium  alcoholate.  This  can  be  done  by  dis- 
solving adenin  in  alcoholic  sodium  hydrate  and  then  adding  methyl 
iodid  and  allowing  to  stand  for  some  months,  or  better,  by  warming 
under  an  inverted  condenser.  It  crystallizes  from  water  in  anhy- 
drous, long,  silky  needles  or  glassy  prisms  resembling  caffein.  On 
drying  in  the  air  the  silky  luster  disappears.  It  may  crystallize  with 
one  and  a  half  molecules  of  water  and  does  not  melt  at  or  below  270°. 
According  to  Fischer,  it  melts  at  308°- 310°  (corr.)  without  decom- 
position. It  is  rather  difficultly  soluble  in  water,  equally  so  in  alka- 
lis; and  is  soluble  in  14  parts  of  hot  water.  The  gold  salt  of 
methyl  adenin  crystallizes  in  fine  yellow  lusterless  needles.  The 
platinochlorid  is  rather  difficultly  soluble  in  cold  water  and  crystal- 
lizes in  bright  four-  or  six-sided  rhombic  plates.  A  1  per  cent. 
^Berichte,  30,  2250  ;  31,  104,  112. 


364  CHEMISTRY  OF  THE  LEUCOMAINS. 

aqueous  solution  of  methyl  adenin  gives  reactions  with  picric  acid, 
silver  nitrate,  aramoniacal  silver  solution,  mercuric  chlorid,  copper 
sulphate,  and  sodium  bisulphite.  The  precipitate  by  the  latter  rea- 
gent is  more  soluble  than  that  of  adenin.  Lead  acetate  and  basic 
lead  acetate  give  no  precipitate.  Like  adenin,  it  forms  a  mono-brom 
derivative  (Kriiger).  The  methyl  compound  of  Thoiss  gave  reac- 
tions with  baryta  water,  alcoholic  zinc  chlorid,  mercuric  nitrate,  and 
cadmium  chlorid,  while  basic  lead  acetate  was  without  effect. 

On  decomposition  with  concentrated  hydrochloric  acid  at  180°— 
200°  it  yields  ammonia,  methylamin  (distinction  from  adenin),  car- 
bonic acid,  formic  acid,  and  glycocoll,  according  to  the  equation  : 

C,H,(CH3)N,  +  8H,0  =  3NH3  +  CH3NH2  +  CO2  + 
2H.CO.OH  +  NH2.CH2.COOH. 

Mono-methyl  adenin  methyl  iodid,  C5H^(CH3)N5.CE[3l,  is  ob- 
tained by  the  action  of  methyl  iodid  on  adenin-lead.  It  crystallizes 
from  alcohol  in  coarse,  glassy  small  crystals  which  are  easily  soluble 
in  alcohol  and  in  water;  insoluble  in  ether.  Simple  substitution 
products  do  not  form  readily,  but  rather  addition  products. 

The  7-methyl  adenin  was  prepared  synthetically  by  Fischer.  It 
melts  at  351°  (corr.)  and  sublimes  at  a  higher  temperature.  It  is 
soluble  in  29  parts  of  boiling  water  and  separates  out  on  cooling  as 
a  granular  powder,  only  occasionally  bent  needles  being  present.  It 
is  difficultly  soluble  in  alcohol  and  forms  salts  with  acids  and  metals 
like  its  isomer. 

Both  methyl  adenins  can  be  readily  changed  into  the  correspond- 
ing methyl  hypoxanthins. 

Ethyl  adenin,  Q^J^Q^^l^^ ,  was  prepared  by  Kriiger  according 
to  the  principle  employed  for  the  preparation  of  the  methyl  deriva- 
tive. It  is  easily  soluble  in  water  and  alcohol,  and  its  aqueous  solu- 
tion gives  reactions  with  silver  nitrate,  ammoniacal  silver  nitrate, 
mercuric  chlorid,  copper  sulphate,  and  sodium  bisulphite,  picric  acid, 
platinum  chlorid,  gold  chlorid.  The  sulphate  crystallizes  from  con- 
centrated solutions  in  roundish  masses  of  crystals. 

Iso-amyl  adenin,  C^H^(C5Hjj)]Sr5 ,  was  also  obtained  by  Kriiger  by 
heating  adenin  with  alcohol,  sodium  hydrate,  and  iso-amyl  iodid.  It 
is  easily  soluble  in  alcohol,  acetone,  chloroform,  hot  benzol  ;  diffi- 
cultly soluble  in  ether  and  carbon  disulphid.  The  solubility  in 
water  at  ordinary  temperature  is  1  :1430,  and  is  not  increased  by 
sodium  or  ammonium  hydrate.  It  is  easily  soluble  in  acids.  It 
forms  large  bright  irregular  plates  which  melt  at  148°-150°.  A 
0.7  per  cent,  aqueous  solution  reacts  with  silver  nitrate,  ammoniacal 
silver  solution,  mercuric  chlorid,  picric  acid,  and  gold  chlorid. 
Platinum  chlorid  does  not  give  a  precipitate. 

The  9-phenyl  adenin  has  been  synthesized  by  Fourneau  (1901) 
from  the  corresponding  uric  acid. 


HYPOXANTHIN.  365 

Adenin-hypoxanthin,  C^H.Ng  +  C^H^Np. — The  occurrence  of 
this  compound  was  observed  by  Kossel,  but  it  was  isolated  and 
studied  for  the  first  time  by  Bruhns.  It  can  be  prepared  by  cooling 
a  hot  aqueous  solution  of  equal  parts  of  the  two  bases.  At  first  it 
is  obtained  as  thick,  starch-like,  semi-transparent  masses,  which  later 
in  part  become  white  and  chalky.  By  spontaneous  evaporation  of 
its  solution  in  very  dilute  ammonia  it  forms  pearly  aggregates  of 
very  small  radially  arranged  needles,  which  contain  water  of  crystal- 
lization. These  effloresce  somewhat  and  lose  the  water  at  100°. 
The  compound  is  more  readily  soluble  in  water  than  its  components, 
but  an  exact  determination  of  its  solubility  is  impossible,  inasmuch 
as  the  separation  from  hot  solutions  is  not  completed  for  some 
weeks.  Any  adenin  present  can  be  separated  by  recrystallization. 
It  forms  a  distinct  crystalline  hydrochlorid,  which  should  be  borne 
in  mind  when  examining  microscopically  for  the  two  bases ;  but  the 
combination  is  loose,  since  addition  of  gold  chlorid  brings  down  the 
characteristic  gold  salt  of  adenin.  Ordinarily  it  does  not  form  salts 
with  sulphuric  or  nitric  acid,  but  more  often  is  decomposed  by 
these,  so  that  the  difficultly  soluble  adenin  crystallizes  out.  Once, 
however,  Bruhns  obtained  a  sulphate  which  differed  from  the 
pure  adenin  and  hypoxanthin  sulphates.  This  perhaps  explains 
the  observation  of  Kossel  that  adenin  sulphate  forms  crystals  belong- 
ing to  two  systems.  The  compound  can  be  decomposed  into  its  con- 
stituents by  fractional  crystallization  of  the  sulphate  or  nitrate  ;  but 
better  by  forming  the  picrates,  which  are  very  unequally  soluble  in 
water.  .  The  existence  of  this  compound  undoubtedly  explains  many 
of  the  mistakes  and  discrepancies  concerning  the  properties  of  hypo- 
xanthin, which  it  resembles  more  than  adenin,  and  for  the  same 
reason,  perhaps,  adenin  was  so  often  overlooked. 

Adenin-theobromin,  C,H.N..C^H3NP2- — This  compound  re- 
sembles the  preceding,  and  was  prepared  by  Kriiger  (1896)  from 
tea-extract.  It  is  easily  soluble  in  hot,  more  difficultly  in  cold 
water.  On  recrystallization  from  water  partial  decomposition  takes 
place.  From  aqueous  solution  picric  acid  throws  down  adenin, 
while  theobromin  remains  in  solution.  It  can  be  obtained,  by  crys- 
tallizing an  aqueous  solution  of  equal  molecules  of  the  two  bases,  as 
fine,  long  prisms  of  marked  silky  appearance. 

Hypoxanthin,  C.H^^p,  sometimes  also  known  as  sarcin  or  sar- 
kin,  was  discovered  by  Scherer  (1850)  in  splenic  pulp  and  in  the 
muscles  of  the  heart,  and  was  named  thus  because  it  contains  one 
atom  of  oxygen  less  than  xanthin. 

The  base  has  been  prepared  synthetically  by  Fischer.^  Thus,  tri- 
chlor  purin  with  alkali  yields  di-chlor  hypoxanthin  which  on  reduc- 

1  Berichte,  30,  2226. 


366  CHEMISTRY  OF  THE  LEUCOMAINS. 

tion  yields  hypoxanthin.  It,  as  well  as  its  methyl  derivatives,  may 
be  also  prepared  from  the  synthetic  adenin  and  its  methyl  derivatives 
by  treatment  with  nitrous  acid. 

It  has  been  obtained,  usually  accompanying  adenin  and  guanin, 
from  nearly  all  of  the  animal  tissues  and  organs  rich  in  nucleated 
cells,  i.  e.,  in  nuclein.  It  has  been  found  in  blood  after  death,  but 
not  in  blood  when  flowing  through  the  blood  vessels.  Salomon  has 
recently  shown  it  to  be  a  normal  constituent  of  urine,  present,  how- 
ever, in  an  exceedingly  minute  quantity. 

From  10,000  liters  of  urine  Kriiger  and  Salomon  obtained  8.5  g. 
of  hypoxanthin.     For  the  other  bases  present,  see  xanthin,  p.  389. 

In  the  blood  and  urine  of  leucocythsemic  patients  it  occurs  in  in- 
creased quantity  owing  to  the  abnormally  large  number  of  nucleated 
white  blood  corpuscles  in  circulation  (p.  348).  Bence  Jones  ob- 
served in  the  urine  of  a  boy,  who  about  three  years  before  showed 
the  symptoms  of  renal  colic,  a  deposit  of  characteristic  whetstone- 
like crystals,  resembling  uric  acid,  but  differing  from  the  latter  by 
dissolving  readily  on  the  application  of  heat,  while  from  hydrochloric 
acid  it  crystallized  in  elongated  six-sided  plates.  These  crystals  he 
believed  to  be  those  of  xanthin,  but  Scherer  and  others  considered 
them  to  be  hypoxanthin.  It  is  therefore  quite  possible,  though  very 
rare,  for  this  base  to  form  a  deposit  in  the  urine  and  to  be  con- 
founded in  shape  with  uric  acid.  Thudichum  obtained  it  from 
the  urine  of  persons  sick  with  liver  or  kidney  diseases.  Accord- 
ing to  Jaksch,  it  is  present  in  exudates  and  transudates  with  uric 
acid. 

Among  other  places  it  has  been  found  in  the  brain,  muscle,  serum, 
marrow  of  bones,  kidney,  heart,  spleen,  liver,  peripheral  muscles 
(sarkin  of  Strecker),  in  the  adrenals  (Holm,  Okerblom) ;  in  the 
spawn  of  salmon  (Piccard),  in  the  testicles  of  the  bull  (Salomon),  in 
the  nuclein  of  pus  and  red  corpuscles  (Kossel),  in  developing  eggs, 
and  in  putrefaction  of  albumin  (Salomon).  It  has  also  been  found 
in  the  spores  of  lycopodium,  and  in  the  pollen  of  various  plants,  in 
seed  of  black  pepper,  in  grass,  clover,  oats,  bran  of  wheat,  larvse  of 
ants  ;  in  the  juice  of  potato  (Schulze)  ;  in  certain  wines  (Kayser)  ; 
in  the  aqueous  decoction  of  beer  yeast  (Schiitzenberger)  ;  and  also 
in  the  liquid  in  which  yeast  is  grown  (Bechamp).  In  the  auto- 
digestion  of  yeast  hypoxanthin  and  xanthin  disappear  and  only 
adenin  and  guanin  remain  (Kutscher)  (see  p.  348). 

Demant  has  shown  it  to  be  relatively  abundant  in  the  muscles  of 
pigeons  in  a  state  of  inanition,  while  in  muscles  of  well  fed  pigeons 
it  is  said  to  be  entirely  absent.  Salomon  found  hypoxanthin  and 
xanthin  in  the  cotyledons  of  lupine,  as  well  as  in  the  sprouts  of  malt, 
while  Reinke  and  Rodewald  observed  these  two  bases  together  with 
guanin  in  -^thalium  septicum.  With  adenin,  xanthin,  and  theo- 
phyllin,  it  occurs  in  tea-leaves  (Kossel)  ;  but  Kriiger  (1896)  showed 


HYPOXANTHIN.  367 

that  it  is  present  in  traces,  if  at  all,  when  the  copper  method  is  used. 
In  other  words,  by  the  action  of  nitrous  acid,  even  in  presence  of 
urea,  adenin  is  partially  changed  to  hypoxanthin  when  the  silver 
salts  obtained  by  the  old  method  are  treated  with  nitric  acid.  Balke 
has  found  it  in  malt-sprouts  by  the  copper  method.  In  the  pollen 
of  the  fir  (Pinus  sylvestris)  Kresling  found  hypoxanthin,  xanthin, 
and  guanin,  but  not  adenin.  In  the  seeds  of  Kandia  dumetorura 
Vogtherr  found  hypoxanthin  and  guanin,  but  no  xanthin. 

Hypoxanthin  has  been  extracted  from  the  pancreas.  Adenin  and 
guanin  in  the  pancreas  readily  change  after  death  into  hypoxanthin 
and  xanthin  (Inoko).  Inasmuch  as  in  the  sterile  auto-digestion  of 
yeast,  as  pointed  out  above,  only  adenin  and  guanin  remain  it  would 
seem  as  if  trypsin  readily  destroyed  hypoxanthin  and  xanthin.  On 
the  other  hand,  in  the  presence  of  bacteria,  possibly  owing  to  the 
formation  of  nitrous  acid,  adenin  and  guanin  are  converted,  as  pointed 
out  by  Inoko,  into  hypoxanthin  and  xanthin.  The  otherwise  dis- 
crepant results  are  thus  readily  explained. 

For  the  accidental  formation  of  hypoxanthin  from  adenin  by 
nitrous  acid  in  the  silver  method,  see  page  409. 

When  a  mixture  of  guanin,  xanthin,  and  hypoxanthin  is  allowed 
to  putrefy,  the  bases  decompose  and  disappear  in  the  order  named. 
Hypoxanthin  resists  bacterial  action  the  longest,  and  this  corre- 
sponds with  its  behavior  to  reagents  (Baginsky).  Adenin  during 
putrefaction,  in  the  absence  of  air,  is  converted  into  hypoxanthin 
and  guanin  is  correspondingly  changed  into  xanthin  (Schindler). 
An  amido  group  is,  therefore,  replaced  by  oxygen,  and  probably  goes 
to  form  urea.  This  conversion  is  a  very  important  fact,  since  the 
process  of  putrefaction,  as  Hoppe-Seyler  has  repeatedly  pointed  out, 
is  analogous  to  the  vital  process,  and  the  same  chemical  change  may 
take  place  in  the  animal  organs.  The  same  change  very  probably 
takes  place  in  the  auto-digestion  of  yeast  provided  living  yeast,  and 
especially  bacteria,  are  present.  In  sterile  auto-digestions,  as  car- 
ried on  in  the  presence  of  an  antiseptic,  only  adenin  and  guanin  are 
present  in  appreciable  amount.  Its  formation  from  adenin  can  be 
represented  thus : 

C,H,N,  +  H,0  =  C,H,Np  +  NH3 . 

Hypoxanthin  occurs  frequently  in  plants  together  with  the  other 
members  of  this  group,  namely,  adenin,  guanin,  xanthin  and  its 
methyl  derivatives.  The  widely  distributed  character  of  these  bases 
is  due  to  the  presence  of  a  parent  substance,  viz.,  nuclein,  the  neces- 
sary constituent  of  all  cells  capable  of  development,  which  under 
the  influence  of  acids,  and  probably  likewise  of  cellular  enzymes, 
decomposes  into  the  above  mentioned  bases.  They  may,  therefore, 
be  considered  as  the  first  steps  in  the  retrograde  metamorphosis  of 
nearly  all  of  the  tissues  of  animals  and  plants. 


368  CHEMISTRY  OF  THE  LEUCOMAINS. 

Recent  advances  in  biological  chemistry  have  shown  that  the 
undeveloped  eggs  of  various  insects  and  birds  yield  much  less  xan- 
thin  bodies  (hypoxanthin,  xauthin,  etc.)  on  treatment  with  dilute 
acid  than  do  the  partially  developed  eggs  (Tichomiroff,  Kossel). 
This  is  dependent  upon  the  remarkable  fact  observed  by  Kossel  that 
the  nuclein  of  undeveloped  chicken  eggs  differs  from  the  nuclein  of 
cell  nuclei  and  resembles  that  obtained  from  milk.  For,  while  the 
nuclein  from  the  cell  nuclei  decomposes  into  adenin,  guanin,  hypo- 
xanthin,  etc.,  that  from  undeveloped  eggs  and  from  milk  yields  no 
nitrogenous  bases  on  treatment  with  acids.  But  as  the  egg  develops, 
i.  e.,  the  nucleated  cells  increase  in  number,  this  latter  nuclein  is 
gradually  converted  or  gives  way  to  the  ordinary  cell  nuclein,  and 
hence  it  is  that  the  chick  embryo  yields  guanin,  hypoxanthin,  and 
possibly  adenin. 

Unquestionably,  the  presence  of  hypoxanthin,  etc.,  in  developing 
cells  is  due  to  the  presence  of  the  nuclein  molecule  from  which  it  is 
readily  split  off.  In  muscle,  however,  hypoxanthin  and  xanthin 
appear  to  exist  preformed,  and  bear  no  relation  to  nuclein,  since  they 
are  in  the  free  condition,  and  can  be  extracted  from  the  tissue  by 
water.  This  condition  is  analogous  to  that  observed  in  plants,  such 
as  tea,  and  in  sprouts  and  is  undoubtedly  due  to  an  enzyme  action 
on  nuclein  (p.  348). 

According  to  the  observations  of  Salomon  and  Chittenden,  hypo- 
xanthin is  formed  by  the  digestion  of  blood  fibrin  with  gastric  juice, 
pancreatic  juice,  or  on  heating  with  water  or  dilute  acids.  Egg 
albumin  under  the  same  conditions  does  not  yield  any  hypoxanthin, 
except  when  treated  with  pancreatic  juice.  These  observations  re- 
quire repetition,  inasmuch  as  the  fibrin  used  undoubtedly  contained 
nuclein,  which,  as  we  now  know,  readily  decomposes  under  those 
conditions  into  its  characteristic  nitrogenous  bases. 

Hypoxanthin  can  be  readily  obtained  from  a  number  of  closely 
related  substances.  Thus,  carnin,  by  the  action  of  oxidizing  agents, 
is  converted  into  hypoxanthin.  For  this  reason  Weidel  and  Schiit- 
zenberger  regarded  hypoxanthin  as  derived  from  carnin  and  this 
view  may  be  in  part  correct  for  it  is  not  improbable  but  that  carnin 
which  is  a  purin  base  exists  at  times  in  the  nuclein  molecule  or  is 
formed  during  the  metabolism  of  the  purin  radical. 

Again,  it  can  be  obtained  from  adenin  (p.  353)  by  the  action  of 
nitrous  acid.  The  relation  that  hypoxanthin  bears  to  uric  acid 
had  not  been  definitely  established  until  Kriiger  showed  that 
the  constitution  of  hypoxanthin  was  closely  connected  with  that 
of  uric  acid  and  the  xanthin  compounds.  Streckler's  belief  that 
hypoxanthin  by  oxidation  yields  xanthin,  and  that  uric  acid 
by  reduction  with  sodium  amalgam  ypelds  first  xanthin  and  then 
hypoxanthin,  was  not  confirmed  by  Kossel  or  by  Fischer.  The 
reported   change  of  uric  acid    into  xanthin  and    hypoxanthin   by 


HYPOXANTHIN.  369 

Sundwik  (p.  337)  will  require  confirmation.  Nevertheless,  the  re- 
searches of  Fischer  have  shown  that  at  least  indirectly  the  purin 
bases  may  be  converted  into  uric  acid  and  vice  versa  (p.  337). 

Hypoxanthin  was  once  regarded  as  a  step  lower  than  guanin 
in  the  series  of  nitrogenous  products  of  regressive  metamorphosis, 
and  consequently  was  considered  as  derived  from  guanin.  The 
investigations  of  Kossel,  however,  show  that  it  arises  not  from 
guanin  but  from  adenin.  On  the  other  hand,  guanin  is  to  be  looked 
upon  as  the  source  of  xanthia.  It  is  probable  that  in  the  organism 
it  is  oxidized  as  soon  as  it  is  set  free  from  the  nuclein,  forming  suc- 
cessively xanthin,  uric  acid,  urea,  etc.,  and  the  small  quantity  pres- 
ent in  the  urine  is  all  that  has  escaped  oxidation.  The  fact  that 
hypoxanthin  is  so  widely  distributed  in  the  organism,  and  in  much 
larger  quantities  than  was  formerly  supposed,  shows  that  it  may  con- 
stitute, together  with  the  closely  related  bodies  creatin,  xanthin, 
guanin,  etc.,  a  part  of  the  antecedents  of  urea  and  of  uric  acid. 
This  view  is  furthermore  strengthened  since  hypoxanthin  is  espe- 
cially abundant  in  those  organs  which  are  most  active  in  producing 
metabolic  changes  in  the  body,  viz.,  the  liver  and  spleen.  The  fact 
that  tryptic  ferments  apparently  decompose  hypoxanthin  and  xanthin 
has  been  already  pointed  out  (p.  348).  The  conversion  of  hypo- 
xanthin into  uric  acid  by  an  emulsion  of  liver  cells  has  been  shown 
by  Wiener  (p.  343). 

When  fed  to  dogs,  it  was  observed  that  the  amount  of  hypoxan- 
thin present  in  the  urine  decreased,  and  even  became  less  in  amount 
than  before  the  experiment ;  but,  on  the  other  hand,  the  amount  of 
xanthin  and  especially  of  uric  acid  in  the  urine  was  found  to  have  in- 
creased above  the  normal.  This  shows  that  hypoxanthin  in  the  body 
is  oxidized  probably  first  to  xanthin,  then  into  uric  acid.  According 
to  Robert,  hypoxanthin  is  a  true  muscle  stimulant  (see  p.  345). 

Hypoxanthin  is  a  white,  colorless,  crystalline  powder,  sometimes 
in  part  amorphous  ;  according  to  Bruhns,  pure  hypoxanthin  does 
not  form  floccules  and  bunches  of  microscopic  needles,  but  usually 
coherent  crusts,  which  consist  of  roundish,  sharp-cornered  granules ; 
some  resemble  octahedra.  The  synthetic  hypoxanthin  separates 
from  hot  aqueous  solution  as  a  colorless  crystalline  powder  (Fischer). 
It  is  soluble  in  about  300  parts  of  cold  water  (Strecker),  but  accord- 
ing to  Fischer  the  synthetic  base  is  soluble  at  19°  in  1415  and  at 
23°  in  1370  parts  of  water.  This  agrees  fairly  with  Scherer's  result 
1 :  1090.  The  base  separates  slowly  from  aqueous  solutions,  and 
when  pure  the  solubility,  even  in  the  beginning,  is  less  than  1  :300. 
At  the  end  of  four  days  Bruhns  found  it  to  be  1  :  1880.  It  is  more 
easily  soluble  in  boiling  water  (78  parts  Strecker,  69.5  parts  Fischer), 
and,  on  cooling,  separates  in  the  form  of  white,  crystalline  floccules, 
thus  differing  from  xanthin  which  is  amorphous.  According  to 
Scherer  the  solubility  in  warm  water  is  1  to  180.  The  solubility  in 
24 


370  CHEMISTRY  OF  THE  LEUGOMAINS. 

cold  alcohol  is  very  slight,  about  1  :  1000.  It  dissolves  in  acids  and 
alkalis  without  decomposition,  and  from  solutions  in  the  latter  it 
can  be  precipitated  by  passing  carbonic  acid,  or  by  the  addition  of 
acetic  acid.     The  aqueous  solution  possesses  a  neutral  reaction. 

The  free  base  can  be  heated  up  to  150°  without  suffering  decom- 
position, but  above  this  temperature  it  sublimes,  and  partially  de- 
composes, with  evolution  of  hydrocyanic  acid.  When  heated  with 
potassium  hydrate  to  200°  it  yields  ammonia  and  potassium  cyanid. 
Heated  with  water  to  200°  it  decomposes  into  carbonic  acid,  jformic 
acid,  and  ammonia,  and  in  this  respect  it  agrees  with  adenin  (page 
342).  The  properties  of  Strecker's  sarkin  agree  closely  with  those 
of  adenin-hypoxanthin  ;  and,  inasmuch  as  the  latter  has  been  often 
described  as  hypoxanthin,  it  is  very  desirable  that  the  properties  of 
hypoxanthin  be  redetermined. 

When  evaporated  with  an  oxidizing  agent,  chlorin  water  and 
nitric  acid,  the  residue  is  said  to  give  on  contact  with  ammonia 
vapors  a  rose-red  color  (Weidel  test).  Kossel,  however,  has  shown 
that  this  is  due  to  the  presence  of  xanthin,  and  that  pure  hypoxan- 
thin does  not  give  either  the  murexid  test  or  the  xanthin  reaction. 
According  to  Strecker,  concentrated  nitric  acid  converts  hypoxanthin 
into  a  nitro  compound,  which  in  turn,  by  the  action  of  a  reducing 
agent,  is  changed  into  xanthin.  This  statement  has  not  been  con- 
firmed either  by  Fischer  or  by  Kossel.  It  does  not  give  a  green  color 
with  sodium  hydrate  and  chlorid  of  lime — distinction  from  xanthin 
(page  392).  Potassium  permanganate  when  added  to  an  acid  solu- 
tion of  the  base  is  very  slowly  decolored  and  hence  can  be  used  for 
its  purification  (Kossel). 

Like  adenin  (page  352),  when  evaporated  with  bromin  water  and 
nitric  acid  on  a  water-bath  it  gives  a  residue  which  with  alkalis  turns 
red,  whilst  nitric  acid  alone,  as  given  above,  has  no  effect  (Kossel). 
An  additional  similarity  to  adenin  is  seen  in  its  behavior  to  zinc  and 
hydrochloric  acid.  The  addition  of  sodium  hydrate  after  reduction 
produces  a  red  color  (page  352)  which  is  not  as  pronounced  as  in  the 
case  of  adenin  (Fischer).^ 

For  the  behavior  of  hypoxanthin  and  other  bases  to  DrechsePs 
reaction,  see  p.  353.  With  copper  sulphate  and  sodium  bisulphite  it 
forms  a  whiter,  more  flocculent  precipitate  than  adenin,  soluble  in 
250,000  parts  of  hot  water  (Kruger).  Its  solubility  and  properties 
are  about  the  same  as  those  of  the  adenin  compound.  0.5  per  cent., 
and  even  stronger  solutions,  are  not  precipitated  in  the  cold  by 
copper  sulphate  and  sodium  hyposulphite.  It  is,  however,  precipi- 
tated on  heating,  whereas  uric  acid  is  not.  It  is,  therefore,  possible 
to  separate  uric  acid  from  adenin  and  hypoxanthin  by  precipitating 
the  latter  two  bases  in  hot  solution  with  copper  sulphate  and  sodium 
hyposulphite.  The  method,  however,  is  of  little  practical  value, 
"  BericJUe,  30,  2230,  2241. 


HYPOXANTHIN.  371 

since  uric  acid  can  be  readily  separated  from  these  two  bases  with 
dilute  acids.  The  separation  may  be  useful  for  guanin  and  xanthin, 
which  are  less  soluble  in  dilute  acids,  and  hence  difficult  to  separate 
from  uric  acid.  By  effecting  the  precipitation  in  cold  solution  of 
the  two  bases,  adenin  can  be  separated  from  hypoxanthin. 

With  acids  it  yields  crystallizable  compounds,  and,  like  the  amido 
acids,  it  forms  compounds  with  bases  and  also  with  metallic  salts, 
such  as  silver  nitrate  and  copper  acetate. 

The  hydrochlorid,  C^H^N^CHCl  +  H2O,  crystallizes  in  needles, 
and,  like  the  nitrate  and  sulphate,  it  is  dissociated  on  contact  with 
water.  The  crystalline  form  is  characteristic  and  distinct  from  that 
of  adenin,  as  well  as  adenin-hypoxanthin.  The  nitrate  forms  thick 
prisms  or  roundish  masses  readily  soluble  in  water  and  ammonia. 
Platinum  chlorid  forms  a  yellow  crystalline  double  salt,  having  the 
composition  C^H.Np.HCl.PtCl, . 

It  does  not  form  a  difficultly  soluble  metaphosphate  as  adenin  or 
guanin  (see  p.  355). 

The  picrate  forms  bright  yellow  prisms  easily  soluble  in  hot  water, 
which  solution  is  not  affected  as  that  of  adenin  by  sodium  picrate. 
According  to  WulflP,  it  possesses  the  formula  C5H^Np.CgH2(N02)30H 
-f  HjO.  It  is  obtained  by  addition  of  picric  acid  to  a  solution  of 
adenin,  or  of  sodium  picrate  to  an  acid  solution  of  adenin.  Depend- 
ing on  the  concentration,  it  precipitates  in  greater  or  less  length  of 
time.  It  is  difficultly  soluble  in  cold  water  ;  easily  in  alkaline,  also 
in  ammonia.  The  estimation  of  adenin  or  guanin  by  picric  acid  in 
the  presence  of  hypoxanthin  is  likely  to  give  a  high  result. 

Hypoxanthin-lead  can  be  prepared,  according  to  Kriiger,  by  add- 
ing a  solution  of  hypoxanthin  in  sodium  hydrate  to  a  solution  of  lead 
acetate.     It  is  amorphous. 

Hypoxanthin  silver,  C5H2Ag2N^O.Il20,  forms  an  amorphous 
colorless  precipitate  when  silver  nitrate  is  added  to  an  ammoniacal 
solution  of  the  base.  All  attempts  to  obtain  a  compound  containing 
but  one  atom  of  silver  in  the  molecule,  corresponding  to  the  adenin 
compound  C^H^AgNg,  have  failed.  The  above  compound  was  first 
prepared  by  Strecker,  and  given  the  formula  C5H^N^O.Ag20 ;  but 
the  former  is  preferable,  since  on  heating  at  120°  two  and  a  half 
molecules  of  water  are  lost  and  2C5H2Ag2Np  +  H2O  (Ag  =  60.2 
per  cent.)  results.  At  140°-150°  it  loses  again  in  weight  and  be- 
comes gradually  gray ;  on  exposure  to  air  it  absorbs  moisture.  In 
this  form  hypoxanthin  can  be  estimated  quantitatively ;  the 
presence  of  sodium  picrate  does  not  interfere,  but  chlorids,  etc.,  do. 
It  is  insoluble  in  hot  water.  The  compound  C5ll2Ag2N^0.3H20  is 
obtained  in  the  form  of  microscopic  needles,  by  treating  pure  hypo- 
xanthin silver  nitrate  with  excess  of  aqueous  ammonia.  On  boiling 
with  ammonia  water  it  is  but  slightly  dissolved,  and  appears  to  lose 
slowly  a  part  of  its  water  of  crystallization.     As  a  result  of  the 


372  CHEMISTRY  OF  THE  LEUCOMAINS. 

decomposition  one  half  of  the  hypoxanthin  passes  into  solution,  and 
can  be  recovered  on  boiling  with  addition  of  silver  nitrate  in  the 
crystalline  form ;  or  in  the  cold,  as  the  usual  amorphous  precipitate, 
C,H,Ag,Np.Hp. 

Hypoxanthin  silver  nitrate,  C5H^Np.AgN03  (Ag  =  35.20  per 
cent.),  is  the  best  known  compound  ;  its  formula  was  established  by 
Strecker.  It  is  obtained  by  dissolving  the  above  precipitate,  pro- 
duced by  addition  of  silver  nitrate  to  an  ammoniacal  solution  of  the 
base,  in  hot  nitric  acid,  specific  gravity  1.1  ;  on  cooling  the  hypo- 
xanthin silver  nitrate  crystallizes  in  the  form  of  tufts  of  microscopic 
needles  or  plates.  Heated  at  100°- 120°  it  remains  constant  in 
weight ;  the  quantity  of  silver  present,  when  determined,  is  always 
somewhat  higher  than  the  theoretical,  especially  if  an  excess  of  silver 
nitrate  is  employed  in  the  precipitation.  The  explanation  of  this 
fact  is  probably  that  given  under  adenin,  though  presence  of  silver 
chlorid  may  partly  be  the  cause.  On  treatment  with  ammonia  it 
loses  not  only  nitric  acid,  but  also  half  of  the  hypoxanthin,  and 
C5H2Ag2N^0.3H20  forms.  The  change  takes  place  readily  even  in 
the  cold,  and  if  during  the  digestion  an  excess  of  silver  nitrate  is 
added,  the  hypoxanthin  set  free  is  converted  into  this  compound, 
which  is  wholly  constant  in  composition  compared  with  the  hypo- 
xanthin silver  nitrate.  The  conversion  is  quantitative.  Very  dilute 
hydrochloric  acid,  as  well  as  hydrogen  sulphid,  removes  the  silver 
from  this  compound. 

Hypoxanthin  silver  picrate,  C5H3AgNp.CgH2(N02)30H  (Ag 
=  22.88  per  cent.),  is  gradually  formed  by  adding  silver  nitrate  to 
a  boiling  solution  of  hypoxanthin  picrate.  The  precipitate  is  granular 
and  of  a  lemon-yellow  color,  and  consists  of  aggregations  of  fine,  short 
needles.  It  is  slightly  soluble  in  hot,  insoluble  in  cold  water.  It 
is,  therefore,  applicable  for  a  quantitative  determination  of  the  base. 
Aqueous  ammonia  very  readily  and  completely  removes  the  picric 
acid  from  the  compound,  and  the  residue  is  hypoxanthin  silver, 
which  is  slightly  colored  yellow  by  a  trace  of  picric  acid ;  half  of 
the  hypoxanthin  passes  into  solution.  Nitric  acid  with  difficulty 
converts  it  into  hypoxanthin  silver  nitrate. 

Hypoxanthin  mercuric  chlorid,  CgHgN^OHgCl,  is  obtained  by  add- 
ing an  equivalent  quantity  of  mercuric  chlorid  to  a  boiling  solution 
of  hypoxanthin.  The  precipitate,  which  increases  on  cooling,  is 
crystalline. 

A  second  compound,  CgHgN^OHg^Clg ,  is  produced  by  adding  a 
strong  excess  of  mercuric  chlorid,  in  the  cold,  to  an  aqueous  solu- 
tion of  hypoxanthin.  It  forms  a  heavy  granular  micro-crystalline 
precipitate,  which  contains  some  water  of  crystallization. 

By  boiling  the  preceding  compound  with  just  sufficient  hydro- 
chloric acid  to  effect  complete  solution,  there  is  formed  on  standing 
a  precipitate  of  white  roundish  aggregates  of  leafy  or  needle-shaped 


HYPOXANTHIN.  373 

glittering   crystals    which    have    the    composition    C.H^N^O.HgClj 

The  following  table  of  Bruhns  illustrates  the  analogy  existing 
between  the  mercury  compounds  of  adenin  and  hypoxanthin  and 
similar  derivatives  of  ammonium  : 

Ammonium.  Adknin.  Hypoxanthin. 

NH,HgCl  CsH.NsHgCl  QHsN^OHgCK  +  H,0) 

NH,Hg,Cl,  C^H.N.Hg.Cl,  C5H,N,0Hg,Cl,(  +  H,0) 

(NH3).HgCI,{  }§g^g^j:g|:2^clH,(N0,),0H    C,H,N,OHgCU  +  H,0) 

Hypoxanthin,  as  well  as  guanin  and  xanthin,  forms  readily  soluble 
compounds  with  fixed  alkalis.  From  these  solutions  the  alkali  com- 
pounds tend  to  crystallize  on  gradual  evaporation,  in  rosettes  or 
bundles  of  needles — difference  from  heteroxanthin  and  paraxanthin 
(Salomon).  The  alkali  solutions  of  the  xanthin  bases  are  precipi- 
tated by  carbonic  acid  and  behave  with  acid  salts,  bicarbonates,  and 
ammonium  salts  the  same  as  the  heteroxanthin-sodium  compound. 

According  to  Bruhns,  hypoxanthin  and  uric  acid  are  unaffected  by 
the  action  of  dry  bromin,  even  at  100°,  but  Kriiger  has  shown  this 
statement  to  be  incorrect.  Bromin  has  no  action  on  hypoxanthin  at 
ordinary  temperatures,  but  at  100°  and  as  high  as  150°  the  latter  is 
changed  quantitatively  into  brom-hypoxanthin.  A  dark-red  crys- 
talline mass  is  obtained  which  contains  six  atoms  of  bromin  to  one 
molecule  of  hypoxanthin.  It  is  a  tetra-bromid  of  brom-hypoxanthin 
hydrobromid,  CgHgBrN^O.HBr.Br^ ,  analogous  to  the  similar  com- 
pound of  adenin.  It  loses  bromin  slowly  in  the  cold,  rapidly  at  120°, 
and  brom-hypoxanthin  hydrobromid  remains,  CgHgBrN^O.HBr. 
From  this  salt  the  free  base  can  be  obtained  after  conversion  into 
the  sodium  compound.  For  this  purpose  the  solution  of  the  salt  is 
treated  with  sodium  hydrate  or  carbonic  acid,  or  saturated  direct 
with  sodium  carbonate,  then  concentrated  to  crystallization. 

Brom-hypoxanthin,  CgHgBrN^O  4-  211^0.  This  can  be  prepared 
as  just  described,  or  by  the  action  of  nitrous  acid  on  brom-adenin  at 
70°.  It  forms  a  heavy  powder  of  small,  coarse  crystals  ;  may  form 
spherical  groups  of  long,  hair-like  needles  containing  \\  molecules 
of  water.  It  is  difficultly  soluble  in  water ;  easily  soluble  in  acids 
and  alkalis.  The  aqueous  solution  reacts  strongly  acid.  It  has  the 
properties  of  a  base  and  an  acid,  behaving  with  alkalis  and  alkali 
carbonates  the  same  as  uric  acid,  setting  free  carbonic  acid  from  the 
latter. 

An  aqueous  solution  of  brom-hypoxanthin  reacts  with  silver 
nitrate,  ammoniacal  silver  solution,  mercuric  chlorid,  copper  sul- 
phate, and  sodium  bisulphite.  In  acid  solution  it  is  precipitated 
by  tannic  acid,  phosphotungstic  acid,  phosphomolybdic  acid,  by  basic 
lead  acetate ;  and  is  not  precipitated  by  lead  acetate  or  by  baryta 
water. 


374  CHEMISTRY  OF  THE  LEUCOMAINS. 

On  heating  brom-hypoxanthin  with  sodium  carbonate,  or  by  pass- 
ing carbonic  acid  into  a  solution  of  the  base  in  sodium  hydrate,  the 
sodium  compound  of  brom-hypoxanthin  forms,  CgHgNaBrN^O  + 
2H2O.  It  is  easily  soluble  in  hot  water ;  rather  difficultly  in  cold 
water.  The  solution  has  an  alkaline  reaction.  The  free  base  can 
be  obtained  by  adding  the  calculated  quantity  of  acid.  The  corre- 
sponding barium  compound  is  obtained  by  passing  carbonic  acid 
into  a  solution  of  brom-hypoxanthin  in  barium  hydrate.  On  con- 
centration it  crystallizes  in  fine  white  needles,  and  has  the  compo- 
sition (C5H2BrN^O)2Ba.  The  lead  compound  forms,  on  the  addition 
of  lead  acetate  to  a  solution  of  the  base  in  sodium  hydrate,  as  an 
amorphous  precipitate. 

The  bromin  in  brom-hypoxanthin  is  held  as  firmly  as  that  in  brom- 
adenin.  Thus  it  is  not  affected  by  heating  with  alcoholic  potash  for 
three  hours. 

On  decomposition  of  brom-hypoxanthin  with  hydrochloric  acid 
and  potassium  chlorate  Kriiger  obtained  alloxan  and  urea.  The 
yield  of  alloxan  is  not  greater  than  that  from  adenin,  and  much  less 
than  that  from  xanthin,  due  undoubtedly  to  the  alloxan  nucleus 
splitting  up  into  simpler  bodies  because  of  a  different  arrangement 
of  bonds. 

The  synthetic  dichlorhypoxanthin  was  prepared  by  Fischer  from 
trichlorpurin  (p.  337). 

Benzyl  hypoxanthin,  CgB[3N^O.CH2.CQHg,  was  obtained  by  Thoiss 
by  the  action  of  nitrous  acid  on  benzyl  adenin.  It  forms  a  white 
crystalline  mass  which  under  the  microscope  consists  of  thin  plates. 
It  is  easily  soluble  in  hot  water,  dilute  alcohol,  and  in  acetic  ether ; 
insoluble  in  ether  and  chloroform.  It  melts  at  280°.  It  appears, 
as  Kossel  pointed  out,  that  adenin  and  hypoxanthin  contain  a 
group,  CgH^N^,  which  he  named  adenyl.  This,  it  will  be  seen,  cor- 
responds to  Fischer's  purin. 

Two  of  the  possible  3-methyl  hypoxan thins  are  known.  The  7- 
and  9-methyl  derivatives  can  be  prepared  from  the  corresponding 
compounds  of  adenin  by  the  action  of  nitrous  acid  (Fischer).^ 

1-7-dimethyl  hypoxanthin,  C5H2(CH3)2N^O.  This  compound  was 
prepared  by  Bruhns,  in  the  same  way  as  methyl  adenin,  by  heating 
hypoxanthin  with  alcohol,  sodium  alcoholate,  and  methyl  iodid. 
Fischer  ^  prepared  the  same  derivative  from  dichlorhypoxanthin  and 
from  7-methyl  hypoxanthin.  A  compound  of  dimethyl  hypoxanthin 
with  sodium  iodid,  C5H2(CH3)2Np.NaI  +  SHgO,  formed  first  which, 
crystallized  from  alcohol,  gave  prismatic  crystals,  easily  soluble  in 
water  and  in  hot  alcohol,  insoluble  in  ether.  The  synthetic  hy- 
poxanthin forms  this  same  characteristic  double  salt.  Other  bases 
of  the  uric  acid  group  are  not  known  to  form  similar  compounds. 

^Berichte,  30,  2409  ;  31,  113. 
^Berichte,  30,  2230,  2411. 


HYPOXANTHIN.  375 

Even  caffein,  which  resembles  dimethyl  hypoxanthin,  does  not  enter 
into  such  a  combination. 

The  free  base  is  obtained  by  treatment  of  this  compound  with 
freshly  precipitated  silver  oxid.  It  melts  at  244°- 246°  (Fischer). 
From  chloroform  it  crystallizes  in  fine  silky  needles,  containing  three 
molecules  of  water  of  crystallization.  From  alcohol  it  crystallizes  in 
groups  of  small  pointed  anhydrous  crystals.  It  is  easily  soluble  in 
water  and  chloroform  ;  less  so  in  alcohol.  A  strong  solution  of  the 
base  reacts  with  silver  nitrate,  with  nitric  acid  and  silver  nitrate, 
with  copper  sulphate  and  sodium  bisulphite  (in  the  cold,  not  on 
warming).  Copper  sulphate  and  sodium  thiosulphate  do  not  give  a 
precipitate.  A  one  per  cent,  solution  of  the  base  gives  precipitates 
with  mercuric  chlorid,  platinum  chlorid,  and  gold  chlorid,  but  not 
with  lead  acetate,  basic  lead  acetate,  picric  acid,  and  ammoniacal 
silver  solution. 

On  account  of  the  solubility  of  this  base  in  water  and  chloroform  ; 
the  fact  that  it  is  not  precipitated  by  ammoniacal  silver  solution, 
which  precipitates  all  bases  of  the  uric  acid  group  containing  a  re- 
placeable imido  group ;  also  on  account  of  its  behavior  with  copper 
sulphate  and  mercuric  chlorid,  Kriiger  concluded  that  hypoxanthin 
contained  only  two  imido  groups  capable  of  substitution.  In  addi- 
tion to  these  adenin  contains  a  third  amido  group,  which  is  not  re- 
placeable, and  which  with  nitrous  acid  is  replaced  by  oxygen.  The 
nitrogen  in  position  9,  however,  is  capable  of  forming  methyl  com- 
pounds. 

On  decomposition  with  concentrated  hydrochloric  acid  at  180°- 
200°  it  yields  one  molecule  of  methylamin  and  two  of  ammonia 
and  one  of  sarkosin,  according  to  the  equation  : 

C5H2(CH3)2Np  +  THp  =  2NH3  +  CH3NH2  +  CO2  + 
2HCOOH  +  CH3.NH.CH2.COOH. 

The  isomer  1-9-di-methyl  hypoxanthin  is  probably  formed  from 
9-niethyl  hypoxanthin  (Fischer). 

Diethyl  hypoxanthin  ethyl  iodid,  C,H2(C2H5)2Np.C2H5l.  This 
compound  was  also  prepared  by  Kriiger,  by  heating  hypoxanthin- 
lead  with  ethyl  iodid.  It  crystallizes  from  alcohol  in  beautiful  four- 
sided  glistening  prisms,  which  are  easily  soluble  in  water  and  in  hot 
alcohol ;  insoluble  in  ether. 

Iso-amyl  hypoxanthin,  C5H3(C5lIjj)Np,  was  likewise  prepared 
by  Kriiger  by  heating  hypoxanthin  in  the  presence  of  sodium  hy- 
drate and  alcohol  with  amyl  iodid.  It  forms  six-sided  rhombic 
plates  and  is  easily  soluble  in  chloroform,  difficultly  in  cold  water, 
insoluble  in  ether. 

Di-iso-amyl  hypoxanthin,  CgH2(C5Hjj)2N^O,  is  formed  in  small 
amount  at  the  same  time  as  the  preceding.  It  yields  a  hydrochlo- 
rid  crystallizing  in  small  needles.     The  base  is  set  free  from  solu- 


376  CHEMISTRY  OF  THE  LEUGOMAINS, 

tions  of  the  salt  as  an  oil  which  on  cooling  becomes  crystalline.  The 
formation  of  these  two  compounds  is  analogous  to  the  benzyl  sub- 
stitutions iu  adenin. 

Ethyl  chloro-carbonate,  acting  on  hypoxanthin  in  the  presence  of 
sodium  hydrate,  produces  a  precipitate  which,  recrystallized  from 
hot  water,  forms  elongated  sharp-angled  plates  which  melt  at  185°- 
190°.  It  is  insoluble  or  difficultly  soluble  in  cold  or  hot  alcohol,  in 
ether,  and  in  cold  water ;  easily  soluble  in  hot  water,  in  sodium 
hydrate,  and  in  hydrochloric  acid.  Its  formula  corresponds  to 
CjHjN^O.CO.CjHg .  It  is,  therefore,  considered  by  Kossel  to  be  a 
urethan  of  hypoxanthin. 

Phosphomolybdic  acid  precipitates  hypoxanthin  from  acid  solu- 
tion, and  in  general  the  base  gives  the  ordinary  alkaloidal  reactions. 

It  is  not  precipitated  by  ammoniacal  basic  lead  acetate.  Copper 
acetate  does  not  precipitate  it  in  the  cold,  but  does  on  boiling.  This 
fact  has  been  made  use  of  in  the  isolation  of  hypoxanthin.  Mer- 
curic chlorid,  as  well  as  mercuric  nitrate,  produces  a  flocculent  pre- 
cipitate. 

Altogether,  in  its  behavior  to  reagents  it  resembles  xanthin  to  a 
very  considerable  degree.  The  two  can  be  separated,  however,  by 
the  different  solubilities  of  the  hydrochlorids  in  water,  and  more 
especially  by  that  of  the  silver  salts  in  nitric  acid. 

Physiological  Action. — 25-100  mg.  begin  to  act  on  frogs  in  from 
six  to  twenty-four  hours,  and  produce  increased  reflex  excitability 
and  convulsive  attacks ;  5-100  mg.  are  fatal  (Filehne).  (See  also 
page  345.)  When  injected  subcutaneously  into  hepatotomized  geese  or 
chickens,  or  when  fed  to  chickens,  a  corresponding  increase  in  uric 
acid  secretion  is  observed  (v.  Mach).  A  similar  conversion  of  hypo- 
xanthin into  uric  acid  is  effected  by  emulsions  of  the  liver  of  certain 
mammals  (Wiener,  page  343).  This  conversion  is  analogous  to  that 
observed  by  Stadthagen  in  the  case  of  guanin  (pages  344,  379),  and 
shows  that  in  the  xanthin  bodies  we  may  have  antecedents  of  uric 
acid  apart  from  the  synthesis  of  the  latter  from  ammonia  in  the  liver, 
or  from  the  direct  decomposition  of  nucleins.  The  process  by 
which  this  change  is  effected  is  undoubtedly  one  of  oxidation. 

Guanin,  C^HgNgO,  was  discovered,  in  1844  by  Unger,  as  a  con- 
stituent of  guano,  in  which  it  is  present  in  varying  quantities  accord- 
ing to  the  region  from  which  the  guano  comes.  Thus  the  Peruvian 
guano  is  reported  as  containing  the  largest  proportion  of  this  base, 
and  on  that  account  this  variety  is  employed  when  it  is  desired  to 
prepare  guanin. 

Fischer  accomplished  the  synthesis  of  guanin  by  changing  di-chlor 
hypoxanthin  with  ammonia  into  an  amino-oxychlor  purin  which  on 
reduction  with  hydriodic  acid  gave  guanin.     Its  isomer^  (p.  341), 

^Berichie,  30,  2247. 


GUANIN.  377 

prepared  from  di-chlor  adenin,  resembles  guanin  very  much  and 
inasmuch  as  it  may  be  looked  upon  as  an  oxidation  product  of 
adenin  Fischer  is  inclined  to  believe  that  it  may  be  found  in  the 
animal  body.  From  guanin  it  is  distinguished  by  the  fact  that  the 
sulphate  contains  one  molecule  of  water  which  is  not  driven  off  at 
120°,  and  also  by  not  yielding  guanidin  on  cleavage. 

More  recently  Traube  ^  has  effected  the  synthesis  of  guanin  by 
starting  out  with  guanidin  and  cyanacetic  acid  ethyl  ester.  A 
pyrimidin  body  was  obtained  which  with  formic  acid  gave  the  purin 
base  guanin. 

Guanin  has  been  met  with  in  a  very  large  number  of  tissues,  both 
animal  and  vegetable ;  in  the  liver,  pancreas,  lungs,  retina,  in  the 
thymus  gland  of  the  calf,  and  in  the  testicle  substance  of  the  bull ; 
in  the  scales  of  the  bleak,  and  in  the  swimming  bladder  of  fish,  as 
well  as  in  the  excrements  of  birds,  of  insects,  as  the  garden  spider, 
in  which  it  occurs  with  a  small  quantity  of  uric  acid  (Weinmann), 
and  is  to  be  regarded  as  a  decomposition  product  of  proteids  in  the 
tissues  of  the  spider.  It  is  also  found  in  the  spawn  and  testicle  of 
salmon,  and  Schulze  and  others  have  shown  it  to  be  present  in  the 
young  leaves  of  the  plane-tree,  of  vine,  etc.,  also  in  grass,  clover, 
oats,  as  well  as  in  the  pollen  of  various  plants.  Kresling  found 
it  in  the  pollen  of  the  fir  with  hypoxanthin  and  xanthin.  Guanin 
and  hypoxanthin,  but  no  xanthin,  are  present  in  the  seeds  of 
Randia  dumetorum  (Vogtherr).  Schiitzenberger  isolated  it,  together 
with  hypoxanthin,  xanthin,  and  carnin,  from  yeast  which  had  been 
allowed  to  stand  in  contact  with  water  at  or  near  the  body  tempera- 
ture. On  the  other  hand  in  sterile  auto-digestion  of  yeast  and  of 
pancreas  Kutscher  found  only  adenin  and  guanin ;  in  the  former 
also  the  hexon  bases,  ammonia,  leucin,  tyrosin,  asparaginic  acid  and 
the  base  CgHgN^O^.  As  pointed  out  on  p.  367  in  the  presence  of 
bacteria,  perhaps  owing  to  the  formation  of  nitrous  acid,  the  guanin 
is  changed  to  xanthin.  Levene  obtained  guanin  and  adenin  from  the 
nucleinic  acids  prepared  from  pancreas  and  from  tubercle  bacilli. 

Pathologically,  it  occurs  in  the  muscles,  ligaments  and  joints  of 
swine  suffering  from  the  disease  known  as  guanin  gout.  Normally, 
guanin,  like  adenin,  is  present  in  muscle  tissue  only  in  traces.  It 
has  never  been  found  in  the  urine,  though  xanthin  has  been  mis- 
taken for  guanin  by  some.  In  their  extensive  studies  upon  the 
alloxuric  bases  Kriiger  and  Salomon  failed  to  obtain  guanin  from 
10,000  liters  of  urine.  Possibly  the  base  was  changed  to  xanthin 
by  the  nitrous  acid  formed  in  the  process,  although  this  is  not  the 
case  with  adenin  which  was  found  in  the  urine.  Altogether  the 
presence  of  guanin  in  urine  remains  yet  an  open  question.  In  some 
cases  of  exudates  and  transudates  guanin  is  present  in  considerable 
amounts  (Jaksch). 

1  Ba-ichte,  33,  1371. 


378  CHEMISTRY  OF  THE  LEUCOMAINS. 

The  pancreas  according  to  Hammarsten  yields  a  nucleo-proteid 
which  on  decomposition  gives  chiefly  guanin.  Bang  has  prepared 
a  nucleinic  acid,  presumably  from  this  proteid,  which  on  hydrolysis 
yields  guanin  (34.35  per  cent.)  and  no  other  base.  Glycerin,  phos- 
phoric acid  and  pentose  are  the  other  products.  Should  Bang's  work 
be  confirmed  it  will  go  to  show  that  there  are  several  nucleinic  acids 
in  the  pancreas,  inasmuch  as  adenin  and  traces  of  xanthin  and  hypo- 
xanthin  have  been  obtained  from  this  organ  (Kossel,  Levene). 

As  to  the  origin  of  this  substance  in  the  organism  very  little  has 
been  known  up  to  within  a  few  years,  except  so  far  as  it  has  been 
shown  to  be,  together  with  other  members  of  this  group,  a  transitory 
product  in  the  metabolism  of  nitrogenous  foods  and  tissues.  In  the  case 
of  the  lower  animals  it  is  evidently  the  end-product  of  all  change,  inas- 
much as  it  is  excreted  as  such.  Our  knowledge  as  to  the  immediate 
origin  of  this  and  the  other  allied  bases  has  been  extended  by  the  bril- 
liant researches  of  Kossel  and  his  pupils  on  the  decomposition  prod- 
ucts of  nuclein,  in  which  he  has  shown  that  this  essential  constituent 
of  all  nucleated  cells,  whether  animal  or  vegetable,  decomposes  un- 
der the  action  of  water,  dilute  acids  or  enzymes  into  adenin,  guanin, 
hypoxanthin,  and  xanthin.  We  know  that  the  first  two  bases  are 
readily  converted  by  the  action  of  nitrous  acid  into  the  other  two  ; 
that  is  to  say,  a  NHg  group  in  these  bases  is  replaced  by  an  atom  of 
O — a  change  which  it  is  not  at  all  unlikely  takes  place  in  the  tis- 
sues, perhaps  in  every  cell  nucleus.  That  such  a  change  is  quite 
probable  is  shown  by  the  putrefaction  experiments  of  Schindler, 
whereby  adenin  and  guanin  were  converted  respectively  into  hypo- 
xanthin and  xanthin.  If  this  explanation  is  correct,  then  adenin 
and  guanin  are  primary  transitional  products  between  the  complex 
nucleo-proteid  on  the  one  hand,  and  hypoxanthin  and  xanthin  on 
the  other.  These  two,  in  turn,  may  form  the  connecting  link 
between  the  former  and  the  final  waste  products  uric  acid  and  urea. 

Schulze  and  Bosshard  (1886)  found  in  young  vetch,  clover,  ergot, 
etc.,  a  new  base,  to  which  they  have  given  the  name  vernin.  It  has 
the  formula  CjgH2oNgOg ,  and  is  of  especial  interest  at  this  point, 
since  on  heating  with  hydrochloric  acid  it  apparently  yields  guanin. 
We  have,  therefore,  at  least  two  well  defined  sources  of  guanin,  the 
nucleins  and  vernin. 

Neither  adenin  nor  guanin  occurs  in  normal  muscle  further  than 
in  mere  traces,  a  fact  which  can  only  be  explained  on  the  ground 
that  the  muscle  tissue  is  poor  in  nucleated  cells,  and  hence  in 
nuclein.  Just  as  the  muscle  cell  has  become  morphologically  differ- 
entiated from  the  typical  cell,  it  may  be  looked  upon  also  as  having 
undergone  a  concomitant  chemical  differentiation  (page  349). 

Guanin  and  creatin  apparently  mutually  replace  one  another. 
Thus,  in  the  muscle,  as  just  stated,  guanin  occurs  only  in  traces 
whereas  creatin  is  especially  abundant.     This  may  find  its  explana- 


GUANIN.  379 

tion  in  the  fact  that  both  are  substituted  guanidins.  Creatin  is 
regarded  by  Hoppe-Seyler  as  an  intermediate  product  in  the  forma- 
tion of  urea,  and  a  similar  role  probably  belongs  to  guanin.  It  has 
already  been  pointed  out  (p.  336)  that  the  purin  bases  may  break 
up  in  part  into  pyrimidin  bodies  and  these  in  turn  may  yield  sim- 
pler products.  It  is  therefore  possible  for  guanin  to  thus  yield  crea- 
tinin  which  on  hydration  would  give  creatin.  The  formation  of  the 
latter  from  hexon  bases  such  as  arginin  is  a  like  possibility.  The 
observation  of  Wiener  that  uric  acid  is  destroyed  by  the  muscle 
and  kidney  of  herbivora  (beef,  horse)  and  by  the  liver  of  carniv- 
ora  (dog,  hog)  emphasizes  a  like  action  of  the  muscles  with  respect 
to  the  purin  bases.  From  Stadthagen's  experiments  on  dogs  we 
know  that  guanin  ingested  produces  an  increase  in  the  amount  of 
uric  acid  and  urea  excreted,  and  the  same  is  also  true  of  the  nuclein 
derived  from  yeast.  These  results  have  led  him  to  the  conclusion 
that  in  mammals  uric  acid  is  a  direct,  or  more  or  less  altered  cleav- 
age product  of  proteids,  notwithstanding  the  fact  that  in  birds  it  is 
the  result  of  synthesis  in  the  liver  (page  344). 

Guanin  as  ordinarily  obtained,  when  ammonia  is  added  to  a  solu- 
tion of  a  salt,  forms  a  white  amorphous  powder.  According  to 
Horbaczewski  ^  it  can  be  obtained  in  a  crystalline  form  by  dissolving 
the  powder  in  a  warm  dilute  alkali  (1-2000),  adding  about  one  third 
volume  of  alcohol  and  then  acidulating  with  acetic  acid.  Any  cloudi- 
ness that  forms  is  removed  by  filtration,  after  which  the  clear  filtrate 
is  allowed  to  stand.  In  time  the  guanin  crystallizes  in  rather  large 
roundish  or  irregular  aggregates  which  resemble  those  of  creatinin 
zinc  chlorid  and  consist  of  long  prisms  or  pyramids  microscopically 
quite  unlike  those  of  xanthin.  The  crystals  contain  no  water  of 
crystallization. 

The  free  base  is  insoluble  in  water,  alcohol,  ether,  and  ammonium 
hydrate ;  easily  soluble  in  mineral  acids,  fixed  alkalis,  and  in  excess 
of  concentrated  ammonium  hydrate.  It  can  be  heated  to  above  200° 
without  undergoing  decomposition.  When  evaporated  with  strong 
nitric  acid  it  gives  a  yellow  residue,  and  this  on  the  addition  of 
sodium  hydrate  assumes  a  red  color,  which  on  heating  becomes 
purple,  then  indigo-blue ;  on  cooling  it  returns  to  a  yellow,  passing 
through  purple  and  reddish-yellow  shades,  due,  according  to  v. 
Briicke,  to  absorption  of  water.  This,  the  so-called  xanthin  reaction, 
was  supposed  to  be  due  to  the  formation  of  xanthin  and  a  nitro- 
product.  It  is  given  best  by  guanin,  then  by  xanthin,  and  is  not 
given  by  either  hypoxanthin  or  adenin. 

Nitrous  acid  converts  it  directly  into  xanthin,  thus  : 

C,H,Np  -f  HNO,  =  C,H,Np,  -^  N,  +  H,0. 

This  reaction  is    identical  with    that  of  adenin,  whereby  hypo- 

1  Zeits.  physiol.  Chem.,  23,  226,  1897. 


380  CHEMISTRY  OF  THE  LEUCOMAINS. 

xanthin  is  formed  (see  p.  353).  By  putrefaction  in  the  absence  of 
air  it  forms  xanthin  (Schindler).  The  change  can  be  represented  by 
the  equation  : 

C,H,Np  +  HP  =  C,H,Np,  +  NH3. 

On  oxidation  with  potassium  permanganate  it  yields  urea,  oxalic 
acid,  and  oxy-guanin.  On  heating  with  hydrochloric  acid  and 
potassium  chlorate  it  is  oxidized  to  carbonic  acid,  guanidin,  and 
parabanic  acid,  according  to  the  equation  : 


CO— NH\  H  N^ 

QH^NjO  +  H.O-f  30=  >CO  +  M>C  =  NH-t-CO,. 

CO— NH/  ^«^ 

Parabanic  Acid.  Guanidin. 


This  decomposition,  noted  by  Strecker,  was  repeated  and  confirmed 
by  Fischer  who  was  unable  to  obtain  alloxan  and  urea,  as  in  the  case 
of  xanthin,  but  instead  isolated  parabanic  acid  and  guanidin.  The 
latter  may  be  identified  as  a  picrate  (Emich). 

According  to  Strecker,  a  small  amount  of  xanthin  is  formed  in 
this  reaction,  and  it  is  quite  possible  that  this  base  is  also  formed  on 
oxidation  with  nitric  acid,  especially  if  it  contains  nitrous  acid. 

On  decomposition  with  concentrated  hydrochloric  acid  at  180°- 
200°  Wulff  obtained  cleavage  products  similar  to  those  of  xanthin, 
namely,  ammonia,  glycocoll,  carbonic  acid,  and  formic  acid.  The 
reaction  is  as  follows  : 

C^H^Np  -f  7Hp  =  4NH3  +  C^H^NO^  +  SCO^  +  CUp,. 

Guanin  combines  with  acids,  bases  and  salts.  It  is  a  very  feeble 
base  as  seen  from  the  fact  that  some  of  its  salts,  as  the  hydrochlorid, 
bichromate,  etc.,  readily  dissociate  on  contact  with  water,  especially 
at  higher  temperatures.  On  the  other  hand,  adenin  is  a  much 
stronger  base.  It  unites  with  bases  to  form  crystalline  compounds  ; 
and  with  one  or  two  equivalents  of  acids  to  form  crystallizable  salts. 
Thus,  with  hydrochloric  acid  it  forms  the  two  salts,  C5H5Np.(HCl)2 
and  C,H,N,O.HCl  +  H„0.  Similar  combinations  can  be  obtained 
with  nitric  acid.  The  sulphate,  (C5H5N50)2.H2SO^,  crystallizes  in 
long  needles,  and,  like  the  other  salts,  is  decomposable  by  water.  It 
contains  two  molecules  of  water,  a  fact  which  distinguishes  guanin 
from  its  isomers.  The  iodid,  nitrate,  oxalate,  and  tartrate  are  also 
known. 

The  platinochlorid,  (C5H,Np.HCl)2PtCl,  +  2Hp,  is  readily  ob- 
tained in  a  crystalline  condition.  The  silver  compound  is  soluble  in 
hot  nitric  acid,  and  on  cooling  separates  out  in  fine,  needle-shaped 
crystals,  having  the  composition  CgH^Np.  AgNOj.  For  its  behavior  to 
DrechsePs  reaction,  precipitation  with  copper  solution  in  the  presence 
of  reducing  substances,  see  page  353.  The  compound  forms  as  a  white 
flocculent  precipitate,  which  soon  turns  greenish  and  tends  to  disso- 


QUANIN.  381 

ciate  on  contact  with  water  (Balke).  It  probably  has  the  formula 
CjH.N^O.CQjO.  As  it  has  more  imido  groups  than  adeniu  and  hy- 
poxanthin,  it  is  probable  that  its  solubility  is  less  than  that  of  the 
copper  compound  of  these  bases. 

The  solutions  of  the  hydrochlorid  are  precipitated  by  mercuric 
chlorid  and  nitrate,  potassium  chromate,  potassium  ferricyanid,  and 
by  picric  acid.  Basic  lead  acetate  gives  a  precipitate  only  on  addi- 
tion of  ammonium  hydrate. 

Guanin  bichromate,  (C5H5iS'50)2.H2Cr207 ,  is  in  composition  analo- 
gous to  that  of  adeuin,  and  was  obtained  by  WulflP  by  adding  potas- 
sium bichromate  to  a  hydrochloric  acid  solution  of  guanin.  It 
appears  as  well  formed,  bright,  orange-colored,  elongated,  four-sided 
prisms,  with  truncated  ends.  On  contact  with  water  it  readily  dis- 
sociates, especially  at  100°.  When  heated  above  100°  it  gives  off 
water  whereas  adenin,  which  is  a  stronger  base,  does  not  dissociate 
on  contact  with  water,  and  is  permanent  at  above  100°. 

Guanin  metaphosphate,  CjHjNjO.HPO,  4-  H2O,  has  also  been 
studied  by  Wulff.  It  is  characterized  by  extremely  slight  solubility 
in  water  and  in  dilute  acids.  Pohl  first  observed  that  sodium  meta- 
phosphate gave  with  guanin  hydrochlorid  a  precipitate  insoluble 
in  excess  of  acids,  but  soluble  easily  in  alkalis.  Liebermann  ob- 
tained a  precipitate  by  adding  metaphosphoric  acid  to  a  solution  of 
guanin  in  sodium  hydrate.  The  same  precipitate,  according  to 
Wulff,  forms  when  an  acid  solution  of  guanin  is  treated  with  meta- 
phosphoric acid.  Contrary  to  Liebermann,  the  salt  has  a  defin- 
ite composition,  and  is  not  prone  to  dissociation  as  is  the  case  with 
other  compounds  of  guanin.  The  precipitation  of  guanin  is  so 
complete  that  in  the  filtrate  picric  acid  gives  no  reaction,  and  silver 
nitrate  produces  only  a  slight  flocculent  precipitate.  Inorganic  salts, 
as  magnesium  sulphate,  may  prevent  or  retard  the  precipitation.  It 
forms  an  amorphous,  porcelain-like  mass  which  is  ignited  with  diffi- 
culty. Dried  at  120°  it  still  retains  one-half  molecule  of  water.  In 
the  presence  of  water  at  high  temperature,  as  in  drying,  a  part  of 
the  compound  is  converted  apparently  into  the  orthophosphate,  so 
that  very  accurate  quantitative  results  are  not  possible. 

Liebermann's  view  that  nuclein  is  a  metaphosphate  of  albumin 
containing  mechanical  admixtures  of  metaphosphates  of  xanthin 
bases  has  been  shown  to  be  wrong.  As  additional  evidence  is  the 
fact  that  guanin  metaphosphate  is  very  difficultly  soluble  in  ammo- 
nium hydrate,  while  nuclein  is  extremely  soluble  in  dilute  ammonia. 

Guanin  ferricyanid,  (C.H^N.O),  .H3Fe(CN)g  -f-  8H2O.  This  com- 
pound separates  slowly,  when  potassium  ferricyanid  is  added  to  a  solu- 
tion of  the  hydrochlorid,  as  small,  bright,  brownish-yellow  four- or  six- 
sided  prisms.  At  100°  it  slowly  loses  weight,  and  heating  for  sev- 
eral hours  at  220°- 130°  is  necessary  to  expel  all  the  water.  The 
composition  of  this  salt  is  noteworthy,  for  one  molecule  of  acid  com- 


382  CHEMISTRY  OF  THE  LEUCOMAINS. 

bines  with  four  of  guanin.  The  trivalent  ferricyanic  acid  does  not 
always  exist  as  such  in  combination,  as  in  the  case  of  the  salt  of 
guanin.  On  the  other  hand,  as  pointed  out  by  Wulff,  guanin, 
though  usually  a  monacid  base,  may  unite  with  different  proportions 
of  acid.  Thus  four  nitrates  are  known,  while  in  the  oxalate  and 
tartrate  three  molecules  of  guanin  unite  with  two  molecules  of  acid. 

Guanin  also  forms  a  compound  with  ferrocyanic  acid,  which  ap- 
pears as  almost  colorless  needles. 

Guanin  nitroferricyanid,  (C,H5N,0)2.H2NOFe(CN),  +  IJHp. 
This  is  likewise  obtained  by  adding  sodium  nitroprussid  to  a  solu- 
tion of  guanin  hydrochlorid.  It  forms  large,  glistening,  brick-red, 
four-sided  prisms  with  pointed  ends. 

Potassium  bismuth  iodid  produces  in  even  very  dilute  solution  of 
the  salts  of  guanin  a  precipitate  which  consists  of  fine,  rather  long 
red  needles.  When  dry  it  forms  a  loose,  deep-red  mass.  On  heat- 
ing even  below  100°  water  is  given  off  and  the  color  changes  to  a 
dark  violet.  It  dissociates  readily  on  contact  with  water.  The 
formula  as  determined  by  Wulff  is  C5H5N,O.HI.2Bil3  +  2H2O. 

Guanin  picrate,  C5H5Np.CgH2(N02)30H  +  Hp.  The  reaction 
with  picric  acid  (Capranica)  is  said  to  be  very  characteristic,  and  a 
means  of  distinguishing  this  base  from  xanthin  and  hypoxanthin. 
It  is  best  obtained  by  adding  a  cold,  saturated  solution  of  picric  acid 
or  of  sodium  picrate  to  the  warm  acidulated  solution  of  guanin  when 
a  light,  crystalline  precipitate  forms.  Under  the  microscope  it  appears 
in  pencil-shaped,  fern-like  tufts  of  fine,  orange-yellow  needles,  rarely 
in  bunches  of  large  needles.  Adenin  picrate  has  a  lighter  color. 
This  compound  is  characterized  by  its  crystalline  form  and  its  ex- 
treme insolubility  in  cold  water  and  in  dilute  acids.  Guanin  solu- 
tions, 1 :  30,000,  are  still  precipitated  by  picric  acid,  though  after 
some  time.  When  dry  it  has  a  golden-yellow  color,  felt-like  con- 
eistency,  and  silky  luster.  On  heating  it  becomes  almost  orange-red, 
and  on  cooling  the  original  color  returns.  At  110°  it  loses  water  of 
crystallization,  the  silky  luster,  and  becomes  light-yellow;  at  190° 
it  begins  to  decompose.  It  dissolves  easily  on  warming  in  fixed 
alkalis  and  carbonates.  A  solution  of  guanin-sodium  is  therefore 
precipitated  only  by  excess  of  picric  acid.  It  is  rather  easily  soluble 
in  warm  dilute  acids  ;  difficultly  in  cold  acids.  It  is  dissociated  by 
water,  alcohol,  and  ammonia,  especially  when  warmed  (Wulff). 

Guanin  silver  picrate,  C,H,AgNp.CgH2(N02)30H  -f  Ipip,  is 
thrown  down  from  a  boiling  solution  of  a  guanin  salt,  treated  with 
excess  of  picric  acid,  by  silver  nitrate  as  a  voluminous,  lemon  yellow 
amorphous  precipitate.  It  is  very  difficultly  soluble  in  hot  water, 
almost  insoluble  in  cold  water.  It  tends  to  become  dissociated  on 
contact  with  water — picric  acid  being  removed.  Ammonia  removes 
the  picric  acid  easily  and  completely,  leaving  guanin-silver  (Wulff). 

Guanin  sodium  dissociates  on  contact  with  water. 


OUANIN.  383 

Brom-guauin,  CjH^BrNjO,  was  prepared  by  Fischer  and  Reese  in 
1883,  and  is  analogous  to  brom-caffein,  adenin,  and  hypoxanthin. 
The  hydrochlorid  dissociates  at  ordinary  temperature.  By  the  action 
of  nitrous  acid  it  is  changed  into  brom-xanthin  just  as  brom-adenin  is 
converted  into  brom-hypoxanthin.  On  prolonged  heating  with  strong 
hydrochloric  acid  it  yields  2-amiuo  6.8  di-oxy  purin  (Fischer). 

Acetyl  guaniu,  C^H^NjO.CO.CHg,  was  prepared  by  Wulff  by 
heating  dry  powdered  guanin  with  acetic  anhydrid.  It  forms  small, 
colorless,  silky  needles  which  are  very  difficultly  soluble  in  cold 
water — in  about  4,000  parts ;  more  difficultly  in  cold  alcohol  and 
almost  insoluble  in  ether.  It  is  soluble  in  about  150  parts  of  boil- 
ing water ;  less  easily  in  hot  alcohol.  It  is  easily  soluble  in  dilute 
acids,  alkalis,  and  ammonia,  especially  on  warming.  On  heating 
with  acids  and  alkalis  it  is  completely  saponified.  From  a  solution 
in  cold  dilute  sodium  hydrate  it  is  precipitated  unchanged  by  car- 
bonic acid.  It  is  likewise  unaffected  by  boiling  water.  At  260°  it 
is  apparently  unchanged. 

Propionyl  guanin,  C5H^N50.CO.CIl2.CIl3,  was  also  prepared  by 
Wulff  by  heating  dried  guanin  with  propionic  anhydrid.  Only  a 
small  part  of  the  guanin  enters  into  combination.  It  forms  peculiar 
crystals,  which  under  the  microscope  appear  as  rather  long  plates  or 
scales,  frequently  with  notched  edges.  The  precipitate  is  very  volum- 
inous and  when  dried  forms  a  light,  felt-like,  white  mass  having 
a  mother  of  pearl  luster.  Its  properties  correspond  to  the  acetyl 
compound.     It  is  likewise  apparently  unaffected  at  260°. 

Benzoyl  guanin,  CgH^N^O.CO.CgH, .  Benzoic  anhydrid  reacts  even 
less  energetically  than  propionic  anhydrid  on  guanin,  and  only  a 
very  small  quantity  of  the  ester  forms.  It  appears  as  small  round 
masses,  which  consist  of  fine  stellate  or  bunched  needles.  It  is 
difficultly  soluble  in  hot  water  and  alcohol,  insoluble  in  ether.  At 
very  high  temperature  it  decomposes  with  brown  coloration.  It  is 
rather  resistant  to  the  action  of  boiling  water,  but  is  readily  saponi- 
fied by  hot  dilute  acids.  Attempts  to  obtain  this  compound  by  the 
action  of  benzoyl  chlorid  on  guanin  or  on  guanin-silver  failed. 

Alkyl  derivatives  of  guanin.  Fischer  and  Reese  attempted  un- 
successfully to  prepare  alkyl  derivatives  of  guanin  by  the  action  of 
methyl  iodid  on  the  lead  and  silver  compounds  of  guanin.  Wulff 
endeavored  to  prepare  benzyl  guanin  in  the  same  way  that  Thoiss 
made  benzyl  adenin,  but  failed.  Even  heating  benzyl  chlorid  with 
guanin  in  the  presence  of  sodium  hydrate  failed.  Attempts  to  pre- 
pare a  dimethyl  derivative  were  likewise  resultless.  Recently,  how- 
ever, Fischer  has  been  able  to  prepare  synthetically  the  7-methyl 
and  the  1-7-dimethyl  guanins.  The  former  is  identical  with 
Kriiger  and  Salomon's  epiguanin  which  will  be  next  described.  The 
latter  on  oxidation  yields  methyl  guanidin,  and  corresponds  to 
heteroxanthin. 


384  CHEMISTRY  OF  THE  LEUCOMAINS. 

Ethyl  guanin,  CjH^NgO.CjHg ,  was  obtained  by  Wulff  by  beating 
guanin  with  ethyl  iodid  in  the  presence  of  sodium  hydrate  and 
alcohol.  It  forms  small  needle-shaped  crystals.  When  dry  it 
yields  a  light,  dry  mass.  It  is  difficultly  soluble  in  water,  very 
difficultly  in  alcohol,  easily  in  mineral  acids.  In  general  it  gives 
the  same  reactions  as  guanin.  Thus,  it  yields  a  silver  compound 
difficultly  soluble  in  ammonium  hydrate  ;  a  finely  crystalline  picrate, 
etc.  The  boiling-hot  aqueous  solution  is  precipitated  by  gold 
chlorid.     Apparently  it  is  not  altered  by  heating  at  280°. 

Physiologically  guanin,  like  uric  acid,  is  inert  (Filehne).  This 
may  be  due  to  the  extreme  insolubility  of  the  base  (p.  345). 

Guanin  may  be  readily  prepared  from  Peruvian  guano  by  boiling 
it  repeatedly  with  milk  of  lime  until  the  liquid  becomes  colorless. 
The  residue,  consisting  largely  of  uric  acid  and  guanin,  is  boiled 
with  a  solution  of  sodium  carbonate,  filtered,  and  the  filtrate,  after 
the  addition  of  sodium  acetate,  is  strongly  acidulated  with  hydro- 
chloric acid.  This  precipitates  the  guanin,  together  with  some  uric 
acid.  The  precipitate  is  dissolved  in  boiling  hydrochloric  acid,  and 
the  guanin  then  thrown  out  of  solution  by  the  addition  of  ammonium 
hydrate. 

A  more  convenient  method  of  isolation  of  guanin  from  Peruvian 
guano  is  that  of  Wulff.  The  guano  is  boiled  with  about  5  per  cent, 
sulphuric  acid  for  4—6  hours,  then  cooled,  and  at  once  filtered.  The 
filtrate  is  rendered  alkaline  with  sodium  hydrate  and  again  filtered. 
This  filtrate,  containing  guanin  and  a  little  uric  acid,  is  now  precipi- 
tated with  ammoniacal  silver  solution.  The  voluminous  precipitate, 
after  standing  twelve  hours,  is  transferred  to  a  thick  plaited  filter, 
and  washed  first  with  cold  then  with  hot  water.  While  still  moist 
the  precipitate  is  removed  from  the  filter  and  introduced  gradually 
into  hot,  dilute  hydrochloric  acid.  The  silver  chlorid  is  filtered  off, 
and  the  filtrate  digested  on  a  water -bath  with  animal  charcoal.  The 
clear  solution  is  then  saturated  with  ammonium  hydrate  to  precipi- 
tate the  guanin.  In  order  to  destroy  the  traces  of  uric  acid  which 
accompany  the  guanin  the  precipitate  is  dissolved  together  with  a 
small  amount  of  urea,  in  boiling  20  per  cent,  nitric  acid,  then  set 
aside  to  crystallize.  The  nitrate  of  guanin  is  now  dissolved  in  dilute 
sodium  hydrate,  and  reprecipitated  by  addition  of  ammonium  chlorid, 
thus  removing  any  traces  of  xanthin  that  may  be  present. 

Inasmuch  as  the  guanin  is  present  in  guano  in  combination  partly 
with  calcium,  partly  as  a  nuclein-like  body,  it  is  not  all  set  free  by  a 
single  boiling  with  dilute  acid.  The  extraction  should,  therefore, 
be  repeated  until  it  ceases  to  be  given  off, 

Guanin  is  also  obtained  in  the  decomposition  of  nuclein  with  dilute 
acids,  and  can,  therefore,  be  prepared  from  such  cellular  organs  as 
the  spleen,  pancreas,  etc.  It  can  also  be  prepared  from  auto-digested 
pancreas  or  yeast  (Kutscher). 


OUANIN.  385 

It  should  be  noted  here  that  in  the  decomposition  of  the  mixed 
silver  compounds  with  hydrogen  sulphid  or  ammonium  sulphid 
(Schindler)  the  guanin  often  only  in  part  passes  into  solution  with 
adenin  and  hypoxanthin,  and  the  remainder  is  held  back  in  the  silver 
sulphid  precipitate.  The  latter  should,  therefore,  be  boiled  with 
dilute  hydrochloric  acid,  and  on  saturating  the  filtrate  with  ammonia 
the  guanin  after  a  while  separates  out.  That  portion  of  the  guanin 
which  does  pass  into  solution  with  the  other  two  bases  is  separated 
from  them  by  digestion  with  ammonia  on  a  water-bath.  The  two 
portions  are  then  combined,  tranferred  to  a  filter,  previously  dried 
at  110°,  and  weighed,  washed  well  with  ammonia,  then  dried  and 
weighed. 

Owing  to  the  slight  solubility  of  guanin  picrate  it  has  been  pro- 
posed by  Wulff  as  a  means  for  the  estimation  of  guanin.  For  this 
purpose  the  neutral  or  acid  guanin  solution  is  precipitated  while 
warm,  with  a  sufficient  amount  of  cold  saturated  picric  acid  solution. 
After  standing  twenty-four  hours  the  solution  is  filtered  through  a 
hardened  filter,  and  the  precipitate  well  drained.  It  is  then  washed 
with  one  per  cent,  picric  acid  and  allowed  to  drain,  after  which  it  is 
placed  between  two  watch-glasses,  and  dried  by  slowly  raising  the 
temperature,  finally  for  one  and  a  half  hours  at  110°.  A  deduction 
is  made  for  the  free  picric  acid  by  determining  the  amount  of  water 
in  the  precipitate  from  the  difference  in  the  weights  before  and  after 
drying.  Allowance  should  be  made  for  one  molecule  of  water  of 
crystallization  that  is  driven  off.  A  further  correction  for  the  solu- 
bility of  the  guanin  salt  should  be  made  by  adding  0.0035  for  each 
100  c.c.  of  the  combined  filtrate  and  wash-water.  The  results  thus 
obtained  are  quite  satisfactory.  Bruhns  has  employed  the  picrate  of 
adenin  in  the  estimation  of  adenin.  Xanthin  and  hypoxanthin  were 
supposed  to  yield  soluble  compounds  with  picric  acid,  so  that  either 
adenin  or  guanin,  or  both,  could  be  separated  from  these  bases  in  this 
way.  Wulff,  however,  has  shown  that  when  guanin  is  precipitated 
by  picric  acid  in  the  presence  of  hypoxanthin  some  of  the  latter  is 
also  precipitated,  so  that  it  is  not  possible  to  separate  the  two  bases 
in  this  way.  The  same  is  true  of  adenin  and  hypoxanthin  if  the 
picrate  is  not  filtered  off  until  after  some  hours. 

In  the  separation  of  adenin  and  hypoxanthin  from  guanin  by 
heating  with  ammonium  hydrate,  some  guanin  is  dissolved,  so  that 
the  filtrate  cannot  be  used  with  accuracy  for  the  separation  of  adenin 
from  hypoxanthin  by  Bruhns'  method.  Wulff  has  endeavored  to 
replace  the  ammonia  with  metaphosphoric  acid.  Guanin  is  precipi- 
tated from  feebly  acid  solutions  by  metaphosphoric  acid  almost  com- 
pletely. The  precipitate  filtered  off,  washed  with  cold  water,  dried 
at  110°,  and  weighed  as  C^H^Np.HPOg  +  iHp,  gives  usually 
slightly  low  results,  owing  to  the  difficulty  of  washing  the  precipi- 
tate, and  the  fact  that  the  amount  of  water  retained  in  drying  varies 
25 


386  CHEMISTRY  OF  THE  LEUCOMAINS. 

through  the  partial  conversion  into  orthophosphate.  Where  more 
accurate  results  are  desired  the  precipitate  can  be  transferred  to  a 
Kjeldahl  flask,  and  the  nitrogen  determined.  The  guanin  can  then 
be  calculated  from  the  amount  of  nitrogen  found. 

Although  adenin  is  precipitated  slowly  by  metaphosphoric  acid, 
it  does  not  interfere  with  the  separation  of  guanin,  since  it  is  soluble 
in  large  excess  of  reagent.  Hypoxanthin  does  not  give  rise  to  a 
difficultly  soluble  metaphosphate,  hence  does  not  interfere  with  the 
precipitation  of  guanin.  In  the  filtrate  from  guanin  metaphosphate 
the  hypoxanthin  can  be  determined  directly  by  Bruhns'  hypoxan- 
thin silver  picrate  method,  though  on  account  of  the  excess  of  meta- 
phosphoric acid  in  the  filtrate  it  is  not  to  be  recommended.  It 
would  be  better  to  precipitate  the  filtrate  with  ammoniacal  silver 
solution,  to  decompose  the  silver  salts  with  dilute  hydrochloric  acid, 
and  then  in  the  filtrate  to  separate,  according  to  Bruhns,  the  adenin 
from  the  hypoxanthin.  The  precipitation  of  guanin  in  the  presence 
of  adenin  should  be  carried  out  in  very  dilute  solutions,  and,  as 
stated,  an  excess  of  reagents  should  be  employed.  The  method 
possesses  decided  advantages  over  the  ammonia  method  of  separa- 
tion, owing  to  the  solubility  of  guanin  in  ammonia,  especially  when 
hot.  According  to  Wulfi*,  100  c.c.  of  a  five  per  cent,  solution  of 
ammonia  dissolves  in  the  cold  0.01  g.  of  guanin.  A  volumetric 
method  for  the  estimation  of  guanin  by  means  of  copper  was  sug- 
gested by  Balke. 

Epiguanin,  CgHyN^O,  was  isolated  from  urine  by  Kriiger  and 
Wolff  in  1893,  but  it  was  not  until  five  years  later  that  its  composi- 
tion and  structure  was  definitely  determined  by  comparison  with  the 
synthetic  7-methyl  guanin  ^  (p.  341),  with  which  it  is  identical.  This 
same  base  was  unquestionably  isolated  by  Salomon '^  as  early  as  1884 
from  hog's  urine ;  later  from  ox  urine  and  leukemic  urine.  The 
amount  of  epiguanin  obtained  by  Kriiger  and  Salomon  ^  from  10,000 
liters  of  urine  is  about  the  same  as  that  of  adenin  (3.40  g.)  and  is 
considerably  less  than  that  of  xanthin  and  its  derivatives  (p.  389). 

It  is  important  to  note  that  this  base  is  probably  present  with  adenin 
and  with  xanthin,  1 -methyl  xanthin  and  hypoxanthin  in  the  adrenals 
(Okerblom*).  Kriiger  and  Salomon  incline  to  the  belief  that  epi- 
guanin, like  the  methylated  xan thins,  results  by  the  cleavage  of  higher 
methylated  homologues  of  guanin  which  are  probably  present  in  the 
food  or  else  it  is  a  constituent  of  the  food  and  as  such  passes  through 
the  body  unchanged.  Some  epiguanin  may  unquestionably  be  de- 
rived directly  or  indirectly  from  the  food,  but  the  probable  isolation 
of  epiguanin  and  the  separation  of  1 -methyl  xanthin  from  the  ad- 

1  Berkhte,  30,  2411  ;  31,  544. 
'Zeits.  physiol.  Chem.,  18,  207  (1893);  24,;390. 
»ZeUs.  physiol.  Chem.,  24,  387;  26,  367,  389. 
*Zeits.  physiol.  Chem.,  28,  60,  1899. 


EPIGUANIN.  387 

renals  would  seem  to  indicate  the  existence  of  di-  or  trimethyl  deriva- 
tives of  guanin  in  the  body  as  products  of  nuclein  metabolism.  A 
1-7  dimethyl  guanin  for  example  might  give  rise  to  epiguanin,  which 
in  turn  would  yield  heteroxanthin  (7-methyl  xanthin).  Similarly 
a  1 -methyl  guanin  is  possible,  which  apparently  is  the  source  of 
1-methyl  xanthin.  Finally  paraxanthin  (1-7-dimethyl  xanthin)  may 
in  part  be  directly  derived  from  the  corresponding  guanin  derivative. 
Additional  light  upon  the  origin  of  epiguanin  has  been  gained  by 
the  observation  of  Fischer  (1898),  that  it  arises  from  an  adenin 
derivative  apparently  by  intramolecular  migration. 

The  natural  as  well  as  the  synthetic  base  is  precipitated  from 
concentrated  solution  of  its  salts  by  addition  of  ammonia  and  imme- 
diate cooling  as  whetstone-shaped  crystals.  Heated  rapidly  in  a 
capillary  it  begins  to  color  at  about  390°  and  later  chars  without 
melting.  The  1-7-dimethyl  guanin  melts  without  decomposition  at 
338°- 340°  (Fischer).  Similar  crystals  are  given  by  Balke's 
episarkin,  which  in  many  other  respects  resembles  epiguanin.  Epi- 
sarkin,  however,  is  not  precipitated  by  picric  acid,  but  is  thrown 
down  by  mercuric  chlorid  and  by  ammoniacal  lead  acetate ;  nor  does 
it  give  the  xanthin  reaction.  From  solutions  in  sodium  hydrate  it 
is  precipitated  by  acids  in  the  form  of  splendid,  matted  silky  prisms 
which  if  agitated  in  water  give  a  peculiar  asbestos-like  appearance. 
The  whetstone-like  forms  on  recrystallization  from  water  yield  the 
prismatic  type. 

It  is  difficultly  soluble  in  hot  water  and  in  ammonia ;  almost  in- 
soluble in  alcohol  and  in  cold  water,  a  fact  which  is  of  value  in  the 
isolation  of  the  base.  The  solubility  in  boiling  water  is  1  to  900 
(Fischer).  It  is  easily  soluble  in  dilute  alkali  and  in  hydrochloric 
and  sulphuric  acids,  difficultly  in  dilute  nitric  acid  from  which  the 
nitrate  separates  on  cooling  in  polyhedric  crystals.  The  sulphate 
forms  fine  bent  needles,  which  quickly  change  to  elongated  often 
six-sided  plates.  From  33  per  cent,  sodium  hydrate  solution  on 
cooling  it  crystallizes,  probably  as  a  sodium  compound,  in  broad, 
glistening  pointed  needles.  Carbonic  acid  precipitates  the  free  base 
from  its  solution  in  dilute  alkali  (Fischer). 

The  picrate,  CgH,N,O.CeH2(N02)30H,  is  difficultly  soluble  in 
water  and  is  useful  for  the  identification  of  epiguanin.  It  forms  a 
voluminous  precipitate  which  consists  of  anhydrous,  peculiarly  bent 
prisms  arranged  in  fan-like  bundles,  or  of  rhombic  or  six-sided 
plates.  At  18°  it  is  soluble  in  2708-2732  parts  of  water  (Kriiger 
and  Salomon);  at  16°  it  is  1  to  3049.  It  begins  to  sinter  at  about 
253°  and  decomposes  suddenly  with  evolution  of  gas  at  257°. 
From  a  slightly  alkaline,  warmed  solution  of  the  picrate  the  free 
base  is  precipitated  at  once  upon  the  addition  of  ammonium  chlorid. 

The  platinochlorid  crystallizes  from  concentrated  solution  in  long 
glistening,  six-sided  orange-colored  prisms  which  on  warming  with 


388  CHEMISTRY  OF  THE  LEUCOMAINS. 

the  mother-liquor  change,  like  the  sulphate,  to  a  different  form 
(Fischer).  Gold  chlorid  yields  a  precipitate  of  fine  yellow  needles 
which,  if  dissolved  in  warm  water  and  the  solution  allowed  to 
evaporate  slowly  in  the  cold,  form  macroscopic,  glistening,  polyhedric 
crystals. 

Potassium  bichromate  when  added  to  a  solution  of  the  chlorid 
yields  in  a  short  time  fine,  glistening,  four-sided  yellow  prisms. 

With  silver  nitrate  and  ammonia  the  aqueous  solution  of  the  base 
gives  a  gelatinous  precipitate.  Silver  nitrate  added  to  the  nitric 
acid  solution  of  the  base  gives  a  colorless  amorphous  precipitate 
which  when  recry stall ized  from  hot  dilute  acid  forms  fine  needles 
(Fischer).  Copper  sulphate  and  sodium  bisulphite  in  the  cold  like- 
wise produce  a  gelatinous  precipitate,  which  in  warm  solutions  how- 
ever is  flocculent.  Copper  sulphate  and  sodium  thiosulphate  pro- 
duce in  warm  solutions  a  flocculent  white  precipitate  which  later 
turns  to  brown. 

While  mercuric  chlorid  produces  in  solutions  of  adenin  and  of 
hypoxanthin  (0.1  to  500)  an  immediate  flocculent  precipitate,  with 
epiguanin  a  considerable  excess  of  the  reagent  is  necessary  and  then 
only  a  cloudiness  results. 

An  aqueous  solution  of  the  base  is  not  thrown  down  by  lead 
acetate,  neutral  or  basic  ;  nor  by  lead  acetate  and  ammonia  in  which 
respect  it  behaves  like  adenin  whereas  hypoxanthin  is  completely 
precipitated  by  this  reagent  as  a  heavy,  flocculent  or  gelatinous  pre- 
cipitate. As  Kriiger  and  Salomon  point  out  an  ammoniacal  solution 
of  lead  acetate  may  by  itself  on  standing  give  a  deposit  which  should 
not  be  mistaken  for  a  precipitate  of  the  base. 

On  evaporation  with  concentrated  nitric  acid  it  leaves  a  yellow 
residue  which  with  sodium  hydrate  becomes  orange-red ;  on  heating 
this  becomes  darker  and  even  slightly  violet  (xanthin  reaction). 
Evaporated  with  hydrochloric  acid  and  a  chlorate  it  gives  a  white 
residue  which  with  ammonia  vapors  become  violet  red  (murexid  test). 

With  nitrous  acid  it  readily  yields  heteroxanthin  (7-methyl  xan- 
thin), and  on  cleavage  with  chlorin  it  gives  guanidin  (Fischer  ^). 

Xanthin,  C^H^N^Oj,  was  discovered  by  Marcet  in  1817  in  a 
urinary  calculus  and  since  then  it  has  been  frequently  found  as  the 
only  or  chief  constituent  of  many  calculi.  It  was  not  synthesized 
until  1897  when  Fischer*  prepared  it  in  two  ways  from  tri-chlor- 
purin.  In  1900  Traube^  prepared  it,  as  well  as  guanin,  uric  acid 
and  the  methyl  xanthins,  from  urea  and  cyanacetic  acid. 

Although  obviously  present  in  the  urine  it  was  not  isolated  there- 
from until  very  recently.     It  is  a  normal  constituent  but  is  present 

1  Berichte,  30,  2413. 

^Berichte,  30,  2232  ;  31,  2562. 

^BerichU,  33,  1371,  3035. 


XANTHIN.  389 

only  in  extremely  minute  quantities.  Thus,  from  10,000  liters  of 
urine,  Kriiger  and  Salomon*  obtained  10.11  g.  of  xanthin,  22.35  g. 
of  heteroxanthin,  31.29  g.  of  1-methyl  xanthin,  15.31  g.  of  para- 
xanthin,  8.50  g.  of  hypoxanthin,  3.54  g.  of  adenin  and  3.40  g.  of 
epiguanin.  It  is  evident  from  this  that  xanthin  and  its  methyl 
derivatives  make  up  the  greater  part  of  the  purin  bases  found  in  the 
urine.  Hitherto  these  urinary  bases  have  been  considered  as  meta- 
bolic products  of  the  nuclein  of  the  tissues  but  recent  investigations 
have  demonstrated  that  there  is  another  and  indeed  principal  source 
for  xanthin  and  its  derivatives.  It  is  now  known  that  caffein  and 
theobromin  undergo  cleavage  in  the  body  and  yield  the  less  methyl- 
ated xanthins.  With  the  fact  in  mind  that  a  large  part  of  the  purin 
bases  in  the  urine  are  not  derived  from  the  nuclein  of  the  tissues  but 
owe  their  origin  to  more  complex  bases  in  the  food  it  is  evident  that 
the  determination  of  the  ratio  of  the  nitrogen  of  uric  acid  to  that  of 
the  purin  bases  is  no  criterion  of  cellular  metabolism  unless  the 
presence  of  these  bases  in  the  food  is  first  eliminated. 

During  the  use  of  sulphur  baths,  or  after  the  thorough  applica- 
tion of  sulphur  salves,  the  quantity  of  xanthin  in  the  urine  is  said  to 
be  considerably  increased.  It  is  likewise  more  abundant  in  the 
urine  of  leukemic  patients,  for  the  reasons  already  given  on  p.  348. 
In  small  amounts  of  leukemic  urine  (two  cases)  Salomon,  however, 
was  not  able  to  detect  it  by  means  of  the  sodium  reaction.  It  was 
found  in  one  out  of  four  pneumonic  urines ;  and  in  two  out  of  ten 
normal  urines  from  which  at  times  it  may  be  wholly  absent. 
Baginsky  holds  that  the  amount  of  xanthin  normally  present  in  the 
urine  may  be  increased  tenfold  in  the  case  of  acute  nephritis. 
Bence  Jones  observed  in  the  urine  of  a  child  sick  with  renal  colic  a 
deposit  of  crystals  which  he  considered  to  be  xanthin,  but  other 
observers  are  inclined  to  regard  the  crystals  as  those  of  hypo- 
xanthin. Vaughan  has  reported  the  presence  of  xanthin  in  deposits 
from  the  urine  of  patients  with  enlarged  spleen. 

Xanthin,  like  the  preceding  three  bases,  is  widely  distributed  in 
animal  tissues  and  in  plants.  In  muscle  and  in  the  pancreas  it  was 
first  detected  by  Scherer  (1859)  and  since  then  it  has  been  obtained, 
though  often  in  mere  traces,  from  all  nucleated  tissues.  Thus,  ac- 
cording to  Schindler,  in  the  thymus  gland  and  in  the  spermatozoa  of 
the  carp  there  are  but  very  small  amounts,  if  any,  of  xanthin, 
whereas  the  sarkin  bases,  especially  adenin,  are  present  in  abundance. 
This  observation  has  been  confirmed  by  Inoko.  The  xanthin  bases 
(xanthin  and  guanin)  are  obtained  in  variable  but  greater  amount 
than  the  sarkin  bases  (adenin  and  hypoxanthin)  from  the  pancreas 
and  from  spermatozoa  of  the  bull,  boar  and  salmon.  The  bases  rich 
in  oxygen,  as  hypoxanthin  and  xanthin,  are  more  abundant  (2  to  1) 
than    those  rich  in  nitrogen  (adenin  and   guanin,  Inoko).      Inoko 

^Zeits.f.  physiol.  Chem.,  26,  367,  1898. 


390  CHEMISTRY  OF  THE  LEUCOMAJtNS. 

found  in  the  semen  of  bull  the  four  bases,  adenin,  guanin,  xanthin, 
and  hypoxanthin.  In  the  nucleinic  acid  from  bull's  testicles  6.039 
per  cent,  of  xanthin  was  found  ;  guanin  was  absent.  The  amount 
of  hypoxanthin  was  also  large,  1.96  per  cent,  against  0.736  per 
cent,  of  adenin.  Drechsel  isolated  xanthin  and  cystin  from  the 
liver  of  the  horse.  Unger  and  Phipson  have  extracted  it  from 
guano. 

From  fresh  adrenals  Okerblom  obtained  less  xanthin  than  when 
these  were  digested  for  two  days  at  37°  in  the  presence  of  chloro- 
form. In  the  latter  case  chiefly  xanthin,  some  1-methyl  xanthin 
and  hypoxanthin  and  traces  of  epiguanin  and  adenin  were  found.  On 
the  other  hand  in  auto-digested  pancreas  and  yeast  Kutscher  found 
only  traces  of  xanthin  and  hypoxanthin  (p.  348)  although  Schiitzen- 
berger  found  both  of  these  bases  and  also  carnin.  Salomon  found  it 
among  the  products  of  the  pancreatic  digestion  of  fibrin  but  it  is 
clear  that  the  latter  must  have  contained  an  admixture  of  nuclein 
(p.  368). 

Together  with  hypoxanthin,  guanin,  and  possibly  adenin,  it  occurs 
in  many  plants,  among  which  may  be  mentioned  lupine,  sethalium, 
sprouts  of  malt  (Salomon,  Balke),  tea-leaves  (Baginsky),  gourd  seeds, 
soja  beans,  etc.,  in  sprouts  of  Cicer  arietinum  (Belsung).  Xanthin 
bases  are  found  in  sprouts  of  lupine  and  of  the  gourd ;  with  hypo- 
xanthin and  guanin  it  occurs  in  the  fir,  Pinus  sylvestris.  Its  pres- 
ence in  auto-digested  yeast  has  already  been  referred  to.  As  pointed 
out  above  the  presence  of  xanthin  and  its  derivatives  in  food,  espe- 
cially in  coffee,  tea,  cocoa,  constitutes  the  chief  source  of  xanthin  and 
of  the  allied  bases  in  the  urine. 

Xanthin  on  electrolytic  reduction  yields  desoxyxanthin,  CjHg 
N,0  (Tafel  and  Ach,  1901). 

It  was  held  by  Strecker  that  xanthin  may  be  obtained  by  reduc- 
tion of  uric  acid  with  sodium  amalgam  according  to  the  equation  : 

Uric  Acid.  Xanthin. 

This  view,  however,  was  not  confirmed  by  Fischer  (p.  336).  More 
recently  Sundwik  claims  to  have  effected  the  reduction  of  uric  acid 
to  xanthin  and  to  hypoxanthin  (p.  337).  The  reverse  operation, 
the  conversion  of  hypoxanthin  into  xanthin,  likewise  reported  by 
Strecker,  has  not  been  confirmed  by  Fischer  or  by  Kossel.  It  is, 
therefore,  evident  that  while  these  bodies  apparently  form  a  contin- 
uous oxidation  series  with  uric  acid  as  the  final  product,  and 
although  this  change  undoubtedly  goes  on  in  the  animal  economy, 
yet  all  attempts  to  reproduce  it  in  the  laboratory  by  oxidation  with 
potassium  permanganate  or  with  nitric  acid  have  yielded  only  nega- 
tive results.  Nevertheless,  in  a  more  indirect  way  it  is  possible  to 
convert  uric  acid  into  the  purin  bases  and  vice  versa.     This  was 


XANTHIN.  391 

demonstrated  in  1895  when  Fischer  effected  the  synthesis  of  caffein 
(trimethyl  xanthin)  from  dimethyl  uric  acid ;  also,  the  conversion  of 
theobromin  into  the  same  uric  acid  derivative. 

Xanthin  can  be  obtained  most  conveniently  by  the  action  of 
nitrous  acid  on  guanin.  The  change  may  be  represented  by  this 
equation  : 

C,H,Np  4-  HNO,  =  C,H,Np,  +  N,  +  H.O. 

Guanin.  Xanthin. 

This  reaction,  first  described  by  Strecker  (1858),  and  later  by 
Fischer,^  corresponds  exactly  to  the  one  by  which  Kossel  transformed 
adenin  into  hypoxanthin  (see  page  353). 

By  putrefaction  guanin  is  also  changed  into  xanthin,  probably  be- 
cause of  the  action  of  the  nitrous  acid  elaborated  by  bacteria  (p.  367). 

Gautier,  starting  out  on  the  hypothesis  that  xanthin  is  a  polymeri- 
zation product  of  hydrocyanic  acid,  endeavored  to  prepare  it  directly 
from  this  compound.  Indeed,  he  claimed  to  have  succeeded  in 
effecting  the  synthesis  of  not  only  xanthin,  but  also  of  its  homo- 
logue,  by  simply  heating  hydrocyanic  acid  in  a  sealed  tube  with 
water  and  a  little  acetic  acid,  the  latter  being  added  to  neutralize 
any  ammonia  that  might  form.  He  expressed  the  reaction  as  fol- 
lows : 

IIHCN  +  4H,0  =  C^H.Np,  +  CgHgN.O^  +  SNHj . 

Xanthin.       Methyl  Xanthin. 

Fischer^  on  repeating  this  experiment  failed  to  obtain  xanthin  and 
since  then  Gautier  has  not  demonstrated  the  correctness  of  his 
former  view. 

Xanthin  is  a  white,  granular,  amorphous  body,  and  is  deposited 
from  hot  aqueous  solution  on  cooling  in  colorless  floccules,  or  as  a 
fine  powder,  which  under  the  microscope  is  seen  to  consist  of  rounded 
granules.  According  to  Horbaczewski^  it  can  be  easily  obtained  in 
the  crystalline  form  by  adding  acetic  acid  to  an  alkaline  solution  of 
the  base  after  previously  diluting  with  hot  water  till  the  solution  is 
about  1  to  2000.  The  clear  liquid,  filtered  if  necessary,  yields  in  a 
few  days  a  deposit  upon  the  sides  and  bottom  of  the  dish  of  groups 
of  crystals.  These  are  thin,  large,  glistening,  rhombic  plates.  If  the 
material  is  impure  leucin-like  balls  or  whetstone-shaped  crystals 
may  form.  The  crystals  contain  one  molecule  of  water,  which  is 
not  lost  at  110°,  but  is  given  off  at  125°- 130°.  The  synthetic 
xanthin  forms  a  colorless  crystalline  powder  (Fischer). 

When  occurring  in  calculi,  it  forms  compact,  moderately  hard  yel- 
low, or  brown  fragments  which  on  being  rubbed  with  the  finger- 
nail, assume  a  wax-like  appearance ;  when  isolated  from  the  urine 

^Ann.  d.  Chem.,  215,  309. 

^Berichte,  30,3131;  31,449. 

*ZeU.  physiol.  Chem.,  23,  226  (1897). 


392  CHEMISTRY  OF  THE  LEUCOMAInS. 

it  is  yellowish,  and  even  when  prepared  from  guanin  it  is  still  col- 
ored. The  decoloration  can  be  eifected,  according  to  Balke,  by  con- 
verting the  xanthin  into  the  mono-sodium  compound  which  can  be 
recrystallized  and  then  decomposed  with  acetic  acid.  The  base  is 
thus  obtained  in  snow-white  floccules. 

It  is  difficultly  soluble  in  cold  water  (about  14,000  parts),  alco- 
hol, and  ether ;  somewhat  more  soluble  in  boiling  water  (about  1,200 
parts).  It  is  soluble  in  alkalis  and  alkali  carbonates,  not  bicarbon- 
ates,  and  from  these  solutions  it  is  precipitated  on  neutralization 
with  acids,  or  by  passing  carbonic  acid.  In  warm  ammonia  it  dis- 
solves more  readily  than  does  uric  acid  or  guanin,  and  on  cooling  the 
ammonium  compound  recrystallizes.  The  solubility  in  cold,  even 
strongly  acid  solutions,  is  very  slight  (Wulff).  It  acts  as  a  weak 
base  and  as  a  weak  acid ;  with  salts  of  the  heavy  metals  it  forms 
difficultly  soluble  or  insoluble  compounds.  Its  basic  properties, 
however,  are  weaker  than  those  of  hypoxanthin  or  guanin. 

When  xanthin  is  evaporated  with  nitric  acid  it  leaves  a  lemon- 
yellow  residue  (hence  its  name),  which  is  not  changed  by  ammonium 
hydrate — distinction  from  uric  acid — but  with  potassium  hydrate  be- 
comes yellowish-red,  on  heating  purple-red.  This,  the  so-called  xan- 
thin reaction,  is  not  given  by  hypoxanthin  or  by  adenin.  On  the 
other  hand,  when  evaporated  with  chlorin  water,  or  with  hydro- 
chloric acid  and  a  chlorate,  it  yields  a  residue  which  with  ammonia 
gives  the  murexid  test  (Fischer,^  Kossel). 

When  added  to  a  mixture  of  bleaching  powder  and  sodium  hydrate 
in  a  watch-glass  the  solution  becomes  covered  by  a  dark -green  scum, 
which  changes  to  a  brown,  and  soon  disappears — distinction  from 
hypoxanthin. 

On  heating  xanthin  a  small  portion  volatilizes ;  the  greater  part 
decomposes  into  ammonium  carbonate,  cyanogen,  and  hydrocyanic 
acid.  Heated  at  200°  with  hydrochloric  acid,  it  decomposes  with 
the  formation  of  ammonia,  carbonic  acid,  formic  acid,  and  glycocoU 
(E.  Schmidt,  see  p.  342).  When  bromin  is  allowed  to  act  on 
xanthin,  there  is  formed  a  substitution  compound,  having  the  for- 
mula CjHgBrN^Og.  It  may  also  be  obtained  by  the  action  of  nitrous 
acid  on  bromguanin  (p.  383).  Brom-xanthin  is  easily  prepared  and 
can  be  readily  changed  to  brom-caffein  which  in  turn  can  be  con- 
verted into  ethoxy-  and  hydroxy-caffein  or  into  caffein  itself. 
Obviously  this  is  a  crucial  test  for  the  recognition  of  xanthin 
(Fischer).^  The  synthetic  chlor-xanthin  is  easily  converted  in  like 
manner.  With  potassium  chlorate  and  hydrochloric  acid  it  yields 
alloxan  and  urea  (p.  341). 

Xanthin  is  a  weak  base,  which  dissolves  in  acids  with  the  forma- 
tion of  salts. 

The  hydrochlorid,  CjH^N^Oj.HCl,  is  difficultly  soluble  in  water, 

1  BericUe,  30,  2236. 
^BerichU,  31,  2563. 


XANTHIN.  393 

more  so  than  the  corresponding  salt  of  hypoxanthin,  and  is  deposited 
in  glistening  six-sided  plates,  often  forming  aggregations.  Its  solu- 
tion does  not  precipitate  platinum  chlorid.  The  nitrate  forms  fine 
yellow  crystals  which  when  pure  are  colorless. 

The  sulphate,  C^H^N^O^.H^SO^  -f  H2O,  crystallizes  in  microscopic, 
glistening,  rhombic  plates,  decomposable  by  water. 

With  baryta  water  xanthin  forms  the  difficultly  soluble  compound 
C5H^N^O.,.Ba(OH)2,  which  corresponds  to  the  hypoxanthin  salt 
C5H^Np'Ba(OH)2,  and  to  that  of  guanin. 

On  the  addition  of  a  very  small  amount  of  sodium  hydrate  to 
xanthin  it  dissolves,  and  very  soon  small  white  needles  separate. 
The  crystals  dissolve  in  excess  of  alkali.  This  xanthin-sodium 
compound,  C5H3NaNP2  +  H^O,  is  also  obtained  by  passing  carbonic 
acid  into  an  alkaline  solution  of  xanthin.  It  forms  small  bunched 
needles,  which  are  rather  easily  soluble  in  water,  imparting  an 
alkaline  reaction.  On  the  addition  of  acetic  acid  the  pure  white  base 
is  thrown  down.  The  compound  is  partly  dissociated  by  hot  water, 
and  resembles  the  corresponding  primary  uric  acid  salts.  It  is 
probable  that  xanthin  can  form,  like  uric  acid,  a  soluble  secondary 
salt,  since  with  excess  of  sodium  hydrate  it  forms  a  readily  soluble 
compound  which  probably  contains  two  atoms  of  sodium.  For  the 
reactions  of  this  compound,  see  page  373.  (See  heteroxanthin  and 
paraxanthin.)  It  does  not  give  a  mono-methyl  xanthin  by  heating 
with  methyl  iodid  (Balke).  The  water  of  crystallization  is  expelled 
only  at  190°- 200°. 

From  amraoniacal  solution  silver  nitrate  precipitates  the  colorless 
compound  C5H^NP2.Ag20,  which  is  unaltered  by  short  boiling  and 
is  insoluble  in  ammonia,  but  soluble  in  hot  nitric  acid.  From  the 
nitric  acid  solution,  on  long  standing,  there  separates  the  compound 
C5H^NP2-^g-^C)3,  which,  on  contact  with  water,  decomposes,  giving 
off  nitric  acid.  The  ammoniacal  solution  is  also  precipitated  by 
lead  acetate — separation  from  hypoxanthin — also  by  calcium  and 
zinc  chlorids.  Cupric  acetate  gives  a  precipitate  only  on  boiling. 
The  aqueous  solution  is  not  precipitated  by  lead  acetate,  but  is  by 
phosphomolybdic  and  phosphotungstic  acids,  by  mercurous  and  mer- 
curic salts.  Picric  acid  gives  an  easily  soluble  compound,  which  re- 
sembles that  of  hypoxanthin,  but  differs  from  that  of  guanin.  Xan- 
thin gives  with  a  copper  solution  and  a  reducing  substance  (Drechsel's 
reaction,  see  page  353)  a  milk-white  precipitate  which  eventually 
becomes  bluish-green  (Balke).  Since  xanthin  has  more  imido  groups 
than  adenin  or  hypoxanthin  and  less  than  uric  acid,  it  is  probable 
that  the  solubility  of  the  copper  compound  will  be  between  the 
solubilities  of  the  corresponding  compounds  of  hypoxanthin  and 
uric  acid  ;  that  is,  between  1  :  250,000  and  1  :  360,000,  the  solubili- 
ties respectively  in  hot  water.  The  copper  compound  of  uric  acid  is 
soluble  in  560,000  parts  of  cold  water  (Kriiger). 


394  CHEMISTRY  OF  THE  LEUCOMAINS. 

As  to  the  physiological  relation  of  xanthin  very  little  need  be 
said  (page  369).  It  bears  the  same  relation  to  guanin  that  hypo- 
xanthin  does  to  adenin,  and,  like  the  latter,  is  to  be  looked  upon  as 
an  intermediate  compound,  a  step  lower  than  guanin,  and  nearer  the 
limit  of  oxidation — uric  acid.  It  is  quite  probable  that  in  the  body 
it  is  oxidized  about  as  rapidly  as  it  is  formed.  Like  hypoxanthin, 
it  is  to  be  regarded  as  a  true  muscle  stimulant,  especially  of  the  heart 
(Baginsky).  According  to  Filehne,  it  produces  in  frogs  a  decided 
muscular  rigor  and  paralysis  of  the  spinal  cord.  The  heart  muscle 
is  also  affected,  which  is  not  the  case  with  caffein  or  theobromin. 
The  fatal  dose  is  less  than  one-half  pro  mille.  In  its  action  it  is 
stronger  than  theobromin,  while  caffein  is  weaker  than  either  of  the 
two.     Paschkis  and  Pal  hold  that  the  reverse  is  true. 

As  pointed  out  on  p.  339  xanthin  may  form  three  mono-methyl  de- 
rivatives— 1-,  3-,  and  7-methyl  xan thins  ;  likewise  three  di-methyl 
compounds,  namely,  theobromin,  theophyllin  and  paraxanthin ;  and 
one  tri-methyl  derivative,  which  is  caffein.  With  the  probable  ex- 
ception of  caffein,  all  of  these  methyl  xanthin  derivatives  appear  in 
the  urine,  where  they  are  to  be  looked  upon  not  as  cleavage  products 
of  tissue  nuclein,  but  rather  as  hydrolytic  bodies  derived  from  the 
caffein  and  other  higher  methyl  derivatives  present  in  the  food. 
For  the  existence  of  other  methyl  derivatives  see  page  340. 

1-Methyl  xanthin,  CgHgNp^?  ^^re  isolated  from  urine  in  1897 
by  Kriiger  and  Salomon.^  As  shown  on  page  389,  it  is  by  far  the 
most  abundant  of  the  purin  bases  in  urine.  Okerblom  has  also  ob- 
tained this  base  from  adrenals  (p.  390),  which  fact  demonstrates  that 
1-methyl  xanthin  may  be  derived  from  higher  homologues  of  xanthin 
contained  within  the  tissue  nuclein,  as  well  as  from  those  contained 
in  the  food.  As  shown  below  it  appears  in  rabbits'  urine  after  feed- 
ing with  paraxanthin.  The  constitution  of  this  base  cannot  be  said 
to  be  definitely  established.  The  conclusion  as  to  its  structure  was 
reached  by  comparison  with  the  3-  and  7-raethyl  xanthins,  with 
neither  of  which  it  was  identical.  Hence  it  was  given  the  structure 
assigned.     The  base  has  not  been  synthesized. 

On  treatment  with  methyl  iodid  it  is  easily  changed  into  caffein 
and  into  theophyllin  (1-3-di-methyl  xanthin)  which  can  be  identi- 
fied by  the  sodium  salt  which  is  rather  difiicultly  soluble  in  ten  per 
cent,  sodium  hydrate  (Kriiger^). 

The  free  base  crystallizes  best  from  acetic  acid  forming  very  thin, 
superposed,  six-sided,  rarely  four-sided  rhombic  plates.  It  separates 
from  water  as  dull,  colorless  crystalline  powder  which  shows  rosette- 
like groups. 

It  is  difficultly  soluble  in  cold  water,  but  more  easily  than  xanthin. 

»  Zeits.  physiol.  Chem.,  24,  380  ;  26,  367. 
'Beriehte,  33,  3666  (1900). 


METHYL  XANTHIN.  395 

It  is  easily  soluble  in  ammonia  and  in  sodium  hydrate,  and  unlike 
heteroxanthin  it  forms  an  easily  soluble  sodium  compound.  The 
barium  salt  unlike  that  of  3-methyl  xanthin  is  very  soluble.  Unlike 
3-methyl  xanthin  it  is  easily  soluble  in  dilute  mineral  acids  and 
from  these  solutions  on  slow  evaporation  in  the  cold  the  correspond- 
ing salts  are  obtained. 

The  chlorid  forms  beautiful  glassy  rhombic  plates  or  prisms.  The 
nitrate  forms  long  four-sided  prisms  at  times  shortened  to  resemble 
six-sided  plates.  These  salts  like  those  of  xanthin  and  heteroxanthin, 
are  easily  dissociated  by  water. 

The  aurochlorid  forms  glistening  rhombic  prisms.  The  platinum 
double  salt  crystallizes  from  concentrated  solutions  in  stellate  need- 
les or  in  prisms. 

With  ammonia  and  silver  nitrate  it  gives  a  gelatinous  precipitate. 
The  silver  nitrate  compound  crystallized  from  dilute  nitric  acid 
(1.1  sp.  g.)  forms,  like  xanthin,  fine  needles  grouped  in  rosettes. 
The  solubility  and  crystalline  form  is  such  that  the  two  bodies  can- 
not be  distinguished  and  as  pointed  out  by  Kriiger  and  Salomon  it 
is  probable  that  the  xanthin  as  heretofore  obtained  may  contain  an 
admixture  of  the  methyl  derivative.  Copper  sulphate  and  sodium 
bisulphite  produce  in  the  cold  a  voluminous  precipitate,  and  in 
warm  solutions  a  white  flocculent  deposit.  Copper  sulphate  and 
sodium  thiosulphate  precipitate  only  on  heating.  Mercuric  chlorid 
produces  a  cloudiness  which  disappears  on  heating  ;  but  on  the  addi- 
tion of  soda  a  white  flocculent  precipitate  results. 

On  evaporation  with  concentrated  nitric  acid  it  gives  the  xanthin 
reaction.  With  concentrated  hydrochloric  acid  and  a  chlorate  it 
gives  an  intense  murexid  test.  In  the  latter  test  the  addition  of 
sodium  hydrate  also  gives  a  purple  red  color  which,  when  the  dish 
is  cool,  on  the  addition  of  a  drop  or  two  of  water  turns  to  a  splen- 
did bluish  violet.  The  latter  disappears  on  heating.  The  base 
yields  a  bromin  derivative  which  is  no  longer  basic  and  is  difficultly 
soluble  in  water  and  in  dilute  acids  and  is  not  altered  at  295°.  It 
dissolves  easily  in  alkalis  and  in  ammonia.  The  sodium  salt  crys- 
tallizes in  six-sided  plates. 

When  paraxanthin  or  1-7-di-methyl  xanthin  is  fed  to  rabbits  a 
part  appears  in  the  urine  unchanged  and  in  addition  1-methyl  xan- 
thin is  present  (Kriiger and  Schmidt^).  The  7-methyl  group  there- 
fore is  split  off  in  the  rabbit  and  this  fact  supports  the  view  of  Kriiger 
and  Salomon  that  this  base  in  man  is  derived  from  paraxanthin. 

3-inethyl  xanthin,  CgHgN^j,  has  not  been  obtained  from  the 
urine  of  man ;  possibly  because  the  methyl  group  in  position  3  is 
easily  split  off  in  the  body.  However,  the  line  of  cleavage  depends 
largely  upon  the  species  of  animal.     It  was  first  found  in  the  urine 

^Berichte,  32,  2680  (1899). 


396  CHEMISTRY  OF  THE  LEUCOMAINS. 

of  dogs  after  feeding  with  caffein  (Albanesi/  Kriiger  ^).  Kriiger  and 
Schmidt^  found  it  in  the  dog  and  rabbit  after  feeding  with  theo- 
bromin  (see  p.  406,  caffein). 

Before  its  discovery  in  urine  this  base  had  been  prepared  synthet- 
ically by  Fischer  and  Ach*  out  of  3-methyl  uric  acid  in  the  same 
way  that  theophyllin  was  first  prepared  from  1-3  di-methyl  uric 
acid. 

The  free  base  is  readily  obtained  from  its  salts  which  are  dissoci- 
ated on  the  addition  of  water  or  by  addition  of  ammonia  and  evap- 
oration to  dryness.  It  forms  a  crystalline  powder.  From  boiling 
water  it  crystallizes  in  fine  glistening  needles,  or  in  small  obliquely 
truncated  prisms.  From  solution  in  alkali  it  is  precipitated  by 
addition  of  acetic  acid. 

When  heated  it  turns  yellow  at  about  360°  and  at  higher  temper- 
ature it  gradually  decomposes  without  melting.  It  is  soluble  in  350 
parts  of  boiling  water  (1-200,  Albanesi),  at  18°  it  is  soluble  in  1,110 
parts  of  water  (Albanesi).  It  is  more  difficultly  soluble  in  absolute 
alcohol,  and  still  more  so  in  chloroform  and  in  acetic  ether.  It  is  very 
easily  soluble  in  dilute  alkalis  and  in  ammonia.  Concentrated  alkali 
precipitates  on  cooling  the  sodium  salt  in  the  form  of  very  fine  bent 
needles.  On  boiling  with  baryta  it  forms  a  very  difficultly  soluble 
compound  (1  :  8,924)  which  crystallizes  in  very  thin,  glistening,  six- 
sided  plates.  This  fact  distinguishes  3-methyl  xanthin  from  1 -methyl 
xanthin  just  as  the  solubility  of  the  sodium  salt  distinguishes  it  from 
7-methyl  xanthin. 

The  base  forms  with  mineral  acids  crystallizable  but  unstable 
salts.  The  chlorid  crystallizes  from  warm  acid  solution  in  fine 
needles.  The  iodid  forms  rather  coarse  prisms.  The  nitrate  sepa- 
rates from  warm  acid  solution  (1.16  sp.  g.)  as  coarse  irregular 
crystals,  and  because  of  its  difficult  solubility  it  can  be  used  for  the 
purification  of  the  base  (distinction  from  1 -methyl  xanthin). 

On  the  addition  of  barium  chlorid  to  an  ammoniacal  solution  of 
the  base  a  precipitate  forms  which  when  dry  dissolves  in  480  (516) 
parts  of  boiling  water  ;  in  750  (862)  parts  of  water  at  18°. 

Silver  nitrate  added  to  a  nitric  acid  solution  of  the  base  produces 
a  crystalline  precipitate,  largely  consisting  of  needles  which  redis- 
solve  on  heating  and  on  subsequent  slow  cooling  long,  thin  prisms 
form.  When  silver  nitrate  is  added  to  an  ammoniacal  solution  of 
the  base  it  yields  a  white  amorphous  precipitate  which  is  not  altered 
by  heat.  The  base  is  thrown  down  by  copper  sulphate  and  sodium 
bisulphite. 

On  oxidation  with  chlorin  it  yields,  like  all  xanthins,  the  murexid 
test. 

^Arch.f.  exp.  Path.  u.  Pharm.,  35,  449;  BericfUe,  32,  2280. 
^Berichte,  32,  2818  (1899). 
3  Beiichte,  32,  2677. 
*Bericht€,  31,  1980  (1898). 


HETEROXANTHIN.  397 

On  treatment  with  methyl  iodid  it  can  be  readily  converted  into 
theobromin  and  into  caffein.  Like  heteroxanthin  it  can  be  subjected 
to  electrolytic  reduction. 

According  to  Albanesi  it  is  more  diuretic  than  7-methyl  xanthin 
(heteroxanthin).  When  fed  to  rabbits  it  is  in  part  excreted  as  such 
and  is  unaccompanied  by  xanthin  (Kriiger  and  Schmidt).  The 
greater  portion  is  probably  broken  up  into  products  simpler  than 
the  purins.     (See  also  p.  407.) 

Heteroxanthin  or  7-methyl  xanthin,  CgHgN^j*  was  isolated 
from  urine  in  1884  by  Salomon,  and  again  in  1893  by  Balke.  The 
amount  present  constitutes  about  two-thirds  of  that  of  1 -methyl 
xanthin  (p.  389).  It  is  a  remarkable  fact  that  this  base  occurs  in 
dog's  urine  unaccompanied  by  paraxanthin,  and  the  same  seems  to 
hold  true  for  the  urine  of  leucocythsemic  persons.  Salomon  examined 
the  liver,  muscles,  and  kidneys  of  a  dog,  but  was  unable  to  obtain  any 
heteroxanthin  or  paraxanthin,  and  the  total  amount  of  xanthin  bodies 
present  was  about  normal.  For  that  reason  he  inclined  to  the  belief 
that  these  two  bases  may  possibly  have  their  origin  in  the  kidney. 
A  certain  amount  of  heteroxanthin  may  unquestionably  be  derived 
from  the  corresponding  guanin  derivative — epiguaniu  (p.  386) ;  and  its 
presence  in  normal  dog's  urine  would  indicate  that  it  is  in  part  a 
product  of  the  metabolism  of  nuclein.  The  greater  part  of  the 
heteroxanthin  is  known,  however,  to  result  from  the  cleavage  of 
higher  homologues  present  in  the  food  such  as  caffein  and  theo- 
bromin (p.  406). 

Paraxanthin  and  heteroxanthin  are  not  present  in  the  urine  or 
kidneys  of  the  cow.  Salomon  found  this  base  in  the  urine  of  one  out 
of  two  leuksemic  patients ;  in  one  with  splenic  tumor,  and  three 
times  out  of  ten  normal  urines.  The  amount  of  xanthin  bodies 
present  in  the  urine  is  unaffected  by  phosphorus  poisoning.  Unlike 
the  other  xanthin  bodies,  heteroxanthin  has  not  as  yet  been  isolated 
from  plants,  meat  extract,  or  guano.  Neither  it  or  paraxanthin 
have  been  found  in  bull's  testicles  (Inoko). 

Heteroxanthin  has  been  prepared  synthetically  by  Fischer^  out  of 
theobromin.  With  phosphorus  oxychlorid  this  yields  a  7-methyl 
2-6-dichlorpurin  which  on  heating  with  concentrated  hydrochloric 
acid  gives  heteroxanthin.  With  slight  modification  the  same  method 
yields  paraxanthin,  7-methyl  hypoxanthin,  7-methyl  guanin,  and 
1-7-dimethyl  guanin.  A  similar  transformation  of  theobromin  into 
heteroxanthin  occurs  in  the  animal  body,  the  methyl  group  in  posi- 
tion three  being  usually  least  firmly  held  in  place.  Heteroxanthin 
can  be  readily  methylated  to  theobromin  and  to  caffein. 

Heteroxanthin  forms  a  white  amorphous  powder,  which  some- 
times on  prolonged  contact  with  water  forms  microscopic  crystalline 

1  Berichte,  30,  2400. 


398  CHEMISTRY  OF  THE  LEUCOMAINS. 

tufts.     Balke  also  described  a  crystalline  modification.     The  syn- 
thetic base  forms  a  colorless  indistinctly  crystalline  powder. 

It  is  very  difficultly  soluble  in  cold  water ;  much  more  easily  in 
hot  water,  and  the  solution  thus  obtained  is  neutral  in  reaction. 
According  to  Fischer  the  solubility  in  boiling  water  is  1:142.  Bond- 
zynski  and  Gottlieb  tested  a  heteroxanthin  which  undoubtedly  con- 
tained 3-methyl  xanthin  (p.  406)  and  found  it  to  be  more  soluble 
in  boiling  water  (1-109) ;  in  water  at  18°  the  solubility  was  1  to 
1,592.  It  was  more  difficultly  soluble  in  absolute  alcohol  and  was  in- 
soluble in  chloroform  and  in  ether.  It  is  easily  soluble  in  ammonium 
hydrate  and  like  uric  acid  dissolves  in  piperazin.  In  3.3  per  cent, 
sodium  hydrate  the  solubility  is  about  1  to  2,100  (Kriiger  and  Salo- 
mon). 

When  slowly  heated  it  partially  melts  and  decomposes  at  341°- 
342°  but  on  rapid  heating  it  begins  to  sinter  at  about  360°,  then 
colors  and  finally  melts  at  380°  with  evolution  of  gas  (Fischer). 

The  hydrochlorid  is  characterized  by  its  rather  difficult  solubil- 
ity and  ready  crystallization  (a  distinction  from  the  paraxanthin 
salt).  The  salt  forms  large  colorless  tufts  of  crystals,  which  on 
contact  with  water  soon  lose  their  transparency  and  become  opaque ; 
gradually  their  crystalline  form  disappears,  till  finally  they  com- 
pletely decompose  with  the  formation  of  heteroxanthin.  This  de- 
composition is  hastened  by  warming,  either  with  or  without  addition 
of  ammonia.  The  nitrate  is  more  difficultly  soluble  and  crystallizes 
from  ten  per  cent,  acid  in  bent  rhombic  plates.  Platinum  chlorid 
produces  in  the  hydrochloric  acid  solution  a  precipitate  of  crystalline 
double  salt. 

On  evaporation  with  nitric  acid  on  the  water-bath  (xanthin  reac- 
tion) it  remains  as  a  pure  white  residue,  which  on  contact  with  sodium 
hydrate  develops  only  a  trace  of  reddish  coloration  or  none  at  all. 
WeidePs  test,  with  concentrated  hydrochloric  acid  and  a  chlorate 
(page  392)  produces  a  splendid  red  color,  which  becomes  blue  on 
the  addition  of  sodium  hydrate.  Simple  evaporation  with  chlorin 
water  gives  a  similar  though  not  so  strong  a  color  reaction.  By 
electric  reduction  it  yields  desoxyheteroxanthin,  CgHgN^O  (Tafel 
and  Weinschenk  ^). 

On  heating  with  concentrated  hydrochloric  acid,  or  with  dilute 
sulphuric  acid,  at  180°- 200°  it  decomposes  and  yields  sarko- 
sin  and  other  products,  according  to  the  equation  (Kriiger  and  Salo- 
mon) : 

CjHgN.O^  +  5Hp  =  200^  +  CO  -h  3NH,  -f 
CH3.NH.CH2.COOH. 

On  treatment  with  methyl  iodid  it  yields  caffein. 
Silver  nitrate  produces   in   ammoniacal   as  well  as  in  nitric  acid 
» Berichte,  33,  3369. 


HETEROXANTHIN.  399 

solutions  a  precipitate  which  readily  dissolves  on  warming  in  even 
very  dilute  nitric  acid  ;  from  this  solution,  if  not  too  concentrated, 
the  heteroxanthin  silver  nitrate  compound  crystallizes  in  character- 
istic well  formed  plate-like  prismatic  crystals  which  are  often 
crossed.  They  are  soluble  in  about  2,800  parts  of  nitric  acid  of  1.1 
specific  gravity  (Kriiger  and  Salomon).  Copper  acetate  produces  in 
the  cold,  in  solutions  of  heteroxanthin,  a  clear  green  precipitate.  The 
base  is  also  precipitated  by  phosphotungstic  acid,  and  by  ammoniacal 
basic  lead  acetate.  Picric  acid  does  not  give  a  yellow-colored  pre- 
cipitate in  solutions  of  the  hydrochlorid. 

Mercuric  chlorid  readily  precipitates  heteroxanthin  in  the  form  of 
a  grayish-yellow  compound,  which  on  standing  twelve  or  twenty-four 
hours  becomes  converted  into  pure  white  crystalline  aggregations. 
This  mercuric  compound  can  be  converted  directly  into  the  corre- 
sponding silver  compound  by  the  addition  of  silver  nitrate  and  am- 
monia, as  described  under  paraxanthin.  For  DrechseFs  reaction 
with  copper,  see  page  353.  The  precipitate  that  forms  is  gelatinous, 
milk-white,  but  soon  turns  green,  as  in  the  case  of  guanin  and  xan- 
thin.  The  solubility  of  the  copper  compound  would  be  about  the 
same  as  adenin  and  hypoxanthin,  since  it  has  two  imido  groups. 
The  addition  of  barium  chlorid  to  an  ammoniacal  solution  of  the 
base  gives  a  gelatinous  precipitate  which  is  rather  soluble  in  hot 
water  and  recrystallizes  on  cooling  in  rosettes  or  balls,  (CgH5N^02)2Ba. 

This  base  resembles  paraxanthin  in  its  property  of  yielding  a  diffi- 
cultly soluble  precipitate  with  fixed  alkali.  This  reaction  is  best 
brought  about  by  dissolving  the  heteroxanthin  hydrochlorid  in  warm 
dilute  sodium  hydrate,  when,  on  cooling,  the  corresponding  sodium 
salt  will  crystallize  out  in  oblique-angled  plates  ;  or  clear  long  prisms, 
(CgH5N^02Na  +  5H2O),  Kriiger  and  Salomon.^  Long-pointed  twin 
crystals  are  the  most  characteristic.  The  crystals  show  greater  vari- 
ations in  form  than  those  of  paraxanthin.  The  behavior  of  the 
twin  crystals  with  polarized  light  is  of  service  in  identification. 
These  crystals  dissolve  easily  in  water,  and  on  neutralization  of  the 
solution  with  an  acid  a  dense  pulverulent  precipitate  of  heteroxanthin 
forms.  This  sodium  compound,  as  well  as  that  of  paraxanthin,  is 
permanent,  non-deliquescent ;  on  moderate  heating  becomes  cloudy, 
and  melts  above  300°.  It  is  more  difficultly  soluble  in  water  than 
the  paraxanthin  compound  ;  the  solution  has  an  alkaline  reaction.  It 
is  soluble  in  mineral  acids  and  ammonia ;  the  latter  redeposits  it 
unchanged  on  evaporation.  On  neutralization  of  its  solution  with 
mineral  acids  or  with  lactic,  acetic  or  carbonic  acids,  the  pure  base 
separates  out  as  amorphous  or  crystalloidal  roundish  or  rosette-like 
masses,  while  paraxanthin  under  the  same  conditions  forms  its  charac- 
teristic crystals.  The  sodium  is  also  removed  from  both  compounds 
by  borax,  potassium  bisulphate,  biphosphate  of  sodium,  bisulphite  of 
'  Zeits.  physiol.  Chem. ,  24,  370. 


400  CHEMISTRY  OF  THE  LEUGOMAINS. 

sodium  and  potassium  bicarbonate,  ammonium  bitartrate.  Ammo- 
nium chlorid,  nitrate,  sulphate,  carbonate,  oxalate,  tartrate,  are  trans- 
posed to  form  sodium  salts,  and  the  free  bases  are  thrown  down. 
Similar  decomposition  of  the  sodium  salts  of  the  two  bases  occurs 
when  placed  in  urine  or  in  meat  extract. 

For  illustrations  of  the  sodium  compounds  of  the  two  bases  see 
Virchow's  Archives,  125,  556. 

The  potassium  compounds  of  heteroxanthin  and  paraxanthin  are 
well  crystallized  bodies,  of  high  melting-point,  and  are  more  soluble 
than  the  sodium  compound.  Their  decompositions  are  the  same  as 
those  of  the  sodium  salt. 

The  reaction  with  sodium  is  the  basis  for  Salomon's  method  of 
recognition  of  these  bases  in  small  quantities  of  urine.  The  other 
two  mono-methyl  derivatives  form  soluble  sodium  compounds. 

It  can  thus  be  distinguished  from  paraxanthin,  the  sodium  com- 
pound of  which,  on  similar  treatment,  yields  the  characteristic  crys- 
talline form  of  the  free  base.  This  sodium  reaction,  therefore, 
distinguishes  it  at  once  from  xanthin,  hypoxanthin,  guanin,  and 
paraxanthin.  It  differs  from  the  latter,  as  has  already  been  indicated, 
in  the  solubility  and  amorphous  character  of  the  free  base ;  in  the 
behavior  of  the  hydrochlorid  and  the  sodium  compound,  and  in  not 
giving  a  precipitate  with  picric  acid,  nor  the  characteristic  odor  given 
by  paraxanthin  on  heating. 

The  physiological  action  of  heteroxanthin  has  been  studied  by 
Kriigerand  Salomon  (1895).  Its  action  is  almost  the  same  as  that 
of  paraxanthin  (page  403),  indicating  a  close  chemical  relation.  Its 
action,  however,  is  much  less  intense.  A  dose  two  or  three  times 
greater  than  that  of  paraxanthin  must  be  injected  into  frogs  to  pro- 
duce the  same  symptoms.  It  has  a  local  action  producing  early  con- 
traction and  rigor  of  the  muscles.  Its  general  action  is  seen  in  the 
gradual  or  rapid  paralysis  of  respiration,  according  to  the  dose ;  in 
the  loss  of  motion  in  the  extremities,  and  in  the  decrease  of  re- 
flexes. It  is  not  as  marked  a  diuretic  as  3-methyl  xanthin  (pages 
345,  397). 

Paraxanthin  or  1-7-dimethyl  xanthin  (p.  339),  CyIIg]Srp2  5  was 

first  isolated  from  urine  by  Thudichum  (1879),  who  named  it  uro- 
theobromin.  It  was  again  isolated  in  1883  by  Salomon,  who  has 
since  shown  it  to  be  a  constituent  of  normal  urine,  although  present 
in  exceedingly  minute  quantity.  Thus  from  1,200  liters  of  urine 
only  1.2  grams  (0.0001  per  cent.)  of  this  substance  were  obtained. 
From  10,000  liters  of  urine  the  yield  was  only  15.31  g.,  or  less  than 
the  amount  of  heteroxanthin  (p.  389).  It  was  also  isolated  in  1893 
by  Balke. 

The  first  synthesis  of  paraxanthin  was  effected  by  Fischer,^  who 
^BeHchte,  30,  2408;  31,  2622. 


PARAXANTHIN.  401 

succeeded  in  transforming  theobromin  into  this  base.  Later  with 
Clemm  he  synthesized  it  direct  from  1.7-dimethyl  uric  acid,  which 
with  phosphorus  oxychlorid  yields  chlorparaxanthin. 

The  supposition  of  Fischer  that  paraxanthin  in  the  urine  was  de- 
rived from  caffein  in  the  same  way  that  heteroxanthin  results  from 
theobromin,  i.  e.,  by  the  splitting  off  of  the  methyl  group  in  position 
3  has  been  recently  confirmed.  When  caffein  is  fed  to  a  dog  in  small 
doses  it  is  excreted  as  3 -methyl  xanthin.  If,  however,  large  doses 
of  caffein  are  administered  some  of  the  base  passes  through  un- 
changed. In  addition  the  urine  contains  all  three  dimethyl  xanthins 
(paraxanthin,  theophyllin  and  theobromin)  and  also  3-methyl  xan- 
thin (Kriiger^).  In  view  of  this  fact  and  inasmuch  as  paraxanthin 
has  not  been  met  with  as  a  direct  cleavage  product  of  nuclein  nor 
found  in  the  urine  of  dogs  or  of  leukemic  patients  it  is  evident  that 
this  base  when  present  in  the  urine  owes  its  origin  to  the  caffein  con- 
tained in  coffee  or  tea. 

Paraxanthin  is  obtained  in  colorless,  glassy,  generally  six-sided 
plates  which  are  arranged  in  tufts  or  rosettes.  From  very  concen- 
trated aqueous  solutions  it  crystallizes  in  long,  colorless,  interwoven 
needles  which  on  drying  exhibit  the  silky  luster  of  tyrosin.  The 
crystals  belong  to  the  monoclinic  system,  and  may  crystallize  with, 
as  well  as  without,  water  (Salomon^). 

If  water  is  present,  on  careful  heating  (110°)  the  crystals  lose 
their  brilliancy  and  become  whitish  and  opaque,  and  at  120°- 130° 
the  water  is  completely  driven  off.  The  conditions  under  which 
crystals  containing  water  are  formed  are  not  known,  probably  by 
slow  crystallization,  whereas  rapid  crystallization  from  hot  concen- 
trated solution  yields  the  anhydrous  needles.  At  about  170°- 180° 
sublimation  takes  place.  The  melting-point  is  at  about  284°  (Kossel), 
295°-  296°  (Fischer).  Theophyllin  melts  at  264°,  while  theobromin 
sublimes  without  melting,  and  caffein  melts  at  232°- 233°.  It  can 
be  heated  to  250°  without  melting  or  suffering  any  decomposition, 
but  when  heated  more  strongly  it  gives  off  white  vapors  which 
possess  a  distinct  iso-nitril  odor  ;  at  the  same  time  it  carbonizes  and 
takes  fire.  When  evaporated  with  concentrated  nitric  acid,  as  in  the 
ordinary  xanthin  test,  it  gives  only  a  slight  yellow  residue.  On  the 
other  hand  when  evaporated  with  chlorin  water  or  with  an  acid  and 
a  chlorate  the  dry  residue  on  exposure  to  an  ammoniacal  atmosphere 
under  a  bell-jar,  gives  a  beautiful  rose-red  color  (so-called  WeideFs 
test). 

It  is  difficultly  soluble  in  cold  water  (though  more  easily  than 
xanthin) ;  somewhat  more  readily  soluble  in  hot  water  (24  parts, 
Fischer)  ;  insoluble  in  ether  and  alcohol.     It  is  soluble  in  ammonium 

1  Berichte,  32,  2818. 

^Virchoufs  Archives,  125,554;  Berichte,  16,195;  18,  3406;  Zdts.  physiol.   Chem., 
11,  415  ;  13,  187. 
26 


402  CHEMISTRY  OF  THE  LEUCOMAINS. 

hydrate,  hydrochloric  acid,  and  nitric  acid.       Its  solutions  are  neu- 
tral in  reaction. 

Silver  nitrate  produces  in  nitric  acid,  as  well  as  in  ammoniacal 
solutions,  a  flocculent  or  gelatinous  precipitate,  which  in  concen- 
trated solutions  forms  an  almost  perfect  jelly-like  mass.  This  silver 
precipitate  is  soluble  in  warm  nitric  acid,  from  which  on  cooling  it 
separates  in  white  crystalline  tufts  possessing  a  silky  luster.  On 
decomposition  with  hydrogen  sulphid  the  silver  salt  yields  pure 
paraxanthin.  Picric  acid  produces  in  the  hydrochloric  acid  solution 
a  precipitate  consisting  of  densely  felted  yellow  crystalline  spangles. 
It  is  also  precipitated  by  phosphotungstic  acid  and  copper  acetate ; 
mercuric  chlorid  when  added  in  excess  gives  after  some  time  a  pre- 
cipitate composed  of  a  mass  of  colorless  prisms,  which  are  rather 
difficultly  soluble  in  water ;  easily  in  hot  water.  The  crystals  of 
paraxanthin  mercuric  chlorid  when  moderately  heated  become 
opaque  from  loss  of  water  of  crystallization  ;  at  a  higher  temperature 
they  melt,  undergoing  at  the  same  time  partial  decomposition,  and 
on  strong  heating  they  evolve  disagreeable  nauseating  vapors.  The 
aqueous  solution  of  this  mercuric  double  salt  gives  with  silver  nitrate 
an  abundant  precipitate  of  silver  chlorid,  which  disappears  on  the 
addition  of  ammonium  hydrate,  and  is  replaced  by  the  flocculent 
gelatinous  precipitate  of  silver  paraxanthin.  The  hydrochloric  acid 
solution  of  paraxanthin  crystallizes  with  difficulty  even  when  strongly 
concentrated,  and  on  the  addition  of  platinum  chlorid  it  yields  a 
well  crystallizable  orange-colored  paraxanthin  platinochlorid.  It  is 
not  precipitated  by  basic  lead  acetate  nor  by  mercuric  nitrate. 
Copper  solutions,  in  the  presence  of  reducing  substances,  give  a 
flocculent  milk-white  precipitate,  which  on  washing  turns  greenish 
by  oxidation  (Balke,  p.  353).  Picric  acid  gives  a  yellow  precipi- 
tate in  a  hydrochloric  acid  solution  of  the  base. 

Like  heteroxanthin  it  yields  a  characteristic  difficultly  soluble 
sodium  salt  which  can  be  made  use  of  for  purpose  of  identification 
and  separation.  The  sodium  salt  has  the  formula  C^HyN^OgNa 
+  4H2O.  It  forms,  upon  the  addition  of  sodium  hydrate  to  a  con- 
centrated solution  of  the  base,  a  precipitate  of  long,  glittering,  crys- 
talline spangles  which  under  the  microscope  are  seen  to  consist  of 
delicate  rectangular,  often  longitudinally  striated  plates  which  are 
isolated  or  united  in  tufts.  The  plates  show  double  refraction.  Be- 
sides these  crystals  there  are  also  present  hexagonal  plates  resembling 
cystin.  The  crystals  are  soluble  in  a  little  water,  or  on  warming, 
but  precipitate  again  on  cooling.  Paraxanthin,  although  it  shares 
with  heteroxanthin  the  property  of  forming  a  difficultly  soluble 
compound  with  fixed  alkalis,  however  can  be  distinguished  from  the 
latter  by  neutralizing  with  an  acid  the  solution  of  the  sodium  or 
potassium  compound,  when,  in  the  case  of  paraxanthin,  there  will 
be  obtained  a  precipitate  of  the  characteristic  crystals  of  that  base ; 


PARAXANTHIN.  403 

whereas  heteroxanthin  on  similar  treatment  gives  a  dense  pulveru- 
lent precipitate.     This  reaction  is  not  given  by  theophyllin. 

Like  the  other  xanthin  derivatives  on  treatment  with  methyl 
iodid  it  yields  caffein. 

The  physiological  action  of  paraxan thin  has  been  studied  by  Salomon 
and  by  Schmiedeberg.  Injections  into  the  muscles  of  1-2  mg.  pro- 
duced almost  at  once  a  rigor  ??ior^/s-like  condition  of  the  muscles  af- 
fected, with  diminished  reflex  excitability  without  previous  increase; 
6—8  mg.  introduced  into  the  lymph  sac  bring  on  a  gradual  loss  of  volun- 
tary motion  as  well  as  of  reflex  excitabilit}^ ;  the  rigor  is  more  marked 
in  the  anterior  extremities,  which  have  a  wooden  or  waxy  consistency. 
Dyspncea  is  likewise  an  early  symptom,  but  as  soon  as  rigor  sets  in 
the  respirations  drop  far  below  the  normal  and  may  be  absent  for 
several  minutes.  At  times  the  lungs  are  enormously  dilated,  same 
as  with  theobromin.  The  heart's  action  is  intact  till  the  very  last. 
In  mice  the  reflexes  are  increased  almost  to  a  tetanus.  An  injection 
of  0.2  g.  in  a  500  g.  guinea-pig  produced  convulsions  and  death  in 
half  an  hour.  The  same  dose  introduced  into  the  vein  of  a  rabbit 
had  no  effect.  The  lethal  dose  for  frogs,  subcutaneously,  was  found 
to  be  0.15—0.2  per  cent,  of  the  body-weight — somewhat  lower  than 
that  of  theobromin  and  xanthin.  The  action  of  these  three  bases  is 
very  similar.  They  produce  in  common  the  slow  creeping  move- 
ments, followed  by  cessation  of  spontaneous  muscle  action,  complete 
loss  of  reflex  excitability  without  a  previous  rise.  The  heart's  action 
is  not  affected  till  in  the  latest  stages. 

Kriiger  and  Schmidt  ^  found  that  paraxanthin  when  fed  to  rabbits 
was  excreted  in  part  as  such  and  in  part  as  1-methyl  xanthin. 
From  this  and  similar  experiments  with  theobromin  and  caffein  it 
would  seem  that  at  least  in  the  bodies  of  rabbits  the  methyl  group  in 
position  3  is  most  easily  split  off,  the  7-methyl  group  next  and  lastly 
that  in  position  1.  When,  however,  3-methyl  xanthin  is  fed  to 
rabbits  it  is  in  part  excreted  as  such  but  this  may  be  due  to  the 
rather  large  doses  administered.  For  the  studies  of  Schmiedeberg 
see  page  345. 

According  to  Rachford*  the  xanthin  bodies  are  important  factors 
in  the  causation  of  migraine.  From  the  urine  passed  immediately 
after  an  attack  of  this  disease  he  obtained  an  extract  which  he 
believed  to  contain  relatively  large  quantities  of  paraxanthin,  and  he 
ascribed  the  marked  toxicity  of  such  extracts  to  this  base.  Pfaff, 
however,  failed  to  find  any  increase  of  paraxanthin  and  interpreted 
the  toxicity  of  the  extracts  as  due  to  the  presence  of  ammonium  salts 
resulting  from  the  method  employed.  Our  present  knowledge  regard- 
ing the  origin  of  this  base  may  be  considered  as  definitely  elimiting 
paraxanthin  as  a  possible  etiological  factor. 

^Berichte,  32,  2677. 

^Tram.  Assoc.  Amer.  Physicians,  10,  11,  14,  224  (1899). 


404  CHEMISTRY  OF  THE  LEUCOMAINS. 

Theophyllin  or  1-3-di-methyl  xanthin  (p.  339),  CyHgNp^ ,  was 

isolated  in  1888  by  Kossel  ^  from  the  alcoholic  extract  of  tea  leaves. 
It  has  not  been  met  with  as  a  product  of  nuclein  metabolism  in  the 
body  nor  even  as  a  cleavage  product.  According  to  Albanesi  a  di- 
methyl xanthin,  presumably  theophyllin,  was  present  in  his  urine 
after  taking  caflPein.  This,  however,  has  been  doubted  by  Kriiger.* 
Nevertheless,  that  theophyllin  may  be  present  in  the  urine  is  shown 
from  Kriiger's  own  experiments  with  caffein  on  dogs  (p.  406). 
When  large  doses  were  administered  all  three  di-methyl  xanthins 
were  present  in  the  urine  and  of  these  theophyllin  was  the  most 
abundant.  It  would  seem  therefore  that  in  the  dog,  at  least,  caffein 
undergoes  cleavage,  yielding  as  the  chief  di-methyl  xanthin,  theo- 
phyllin, and  on  further  cleavage  this  yields  3-methyl  xanthin.  In 
other  words  the  methyl  group  in  position  7  is  the  first  to  be  split  off 
and  that  in  position  1  follows  next.  The  large  amounts  of  1-  and 
7-methyl  xanthins  in  the  urine  of  man  would  indicate  that  in  the 
human  body  the  cleavage  is  different  in  kind  from  that  in  the  dog 
and  is  more  nearly  like  that  in  rabbits. 

Theophyllin  was  the  first  xanthin  derivative  synthesized  by 
Fischer  and  Ach.^  1.3-di-methyl  uric  acid  with  phosphorus  oxy- 
chlorid  yields  chlor-theophyllin  which  on  reduction  yields  the  base 
(p.  337).  Traube  *  prepared  theophyllin  as  well  as  the  above  uric 
acid  out  of  di-methyl  urea  and  cyanacetic  acid.  With  methyl  iodid 
it  yields  caffein. 

In  tea  extract  the  base  is  associated  with  caffein,  xanthin,  adenin 
and  hypoxanthin.  Further  investigations  are  necessary  to  determine 
whether  the  other  di-methyl  xanthins  and  the  mono-methyl  xanthins 
are  present  in  the  extract.  The  origin  of  these  xanthin  derivatives 
in  the  tea-plant  is  likewise  involved  in  obscurity.  They  may  be 
regarded  as  direct  synthetic  products  or  as  cleavage  forms  from 
nuclein. 

Inasmuch  as  theophyllin  cannot  be  regarded  as  a  product  of  tissue 
metabolism  a  detailed  description  of  its  properties  in  this  connection 
is  unnecessary.  The  reader  is  therefore  referred  to  KosseFs  original 
paper. 

Theobromin  or  3-7-di-methyl  xanthin  (p.  336),  CyHgN^Og ,  was 
isolated  from  cocoa  in  1842  by  Woskresensky,  but  its  structure  was 
not  determined  until  1883.  It  has  been  found  only  once  in  urine, 
and  then  in  the  case  of  a  dog  after  administration  of  caffein  in  large 
doses  (p.  406).  The  amount  of  theobromin  was  about  the  same 
as  that  of  paraxanthin  and  was  much  less  than  that  of  theophyllin. 

1  Zeit!i.  physiol.  Chem.,  13,  298. 

2  Berichte,  32,  2819,  2280. 
^Benchte,  28,  3135. 

« Berichte,  33,  3052. 


CAFFEIN.  405 

Obviously  theobromin  may  be  expected  under  like  conditions  in 
human  urine. 

The  base  has  been  synthesized  by  Fischer'  out  of  1-3-di-methyl 
uric  acid  ;  also  from  uric  acid  itself  by  converting  this  into  3-methyl 
uric  acid.  Traube^  likewise  prepared  theobromin  by  treating  3- 
methyl  xanthin  with  methyl  iodid.  Theobromin  can  be  readily 
methylated  to  caffein. 

The  changes  which  theobromin  undergoes  in  the  body  have  been  the 
subject  of  a  number  of  very  interesting  investigations.  Bondzynski 
and  Gottlieb^  in  1895  showed  that  it  underwent  cleavage  when 
fed  to  rabbit  and  to  dogs.  In  rabbits  only  19  per  cent,  of  the 
theobromin  was  recovered  unchanged,  and  24.6  per  cent,  was  ob- 
tained as  a  methyl  xanthin  which  these  workers  identified  with 
heteroxanthin,  although  as  pointed  out  on  p.  398  this  substance 
must  have  been  mixed  with  some  3-methyl  xanthin.  Xanthin 
itself  was  not  obtained.  Similar  though  incomplete  experiments 
with  caffein  seemed  to  give  the  same  methyl  xanthin.  They  also 
found  that  when  theobromin  was  administered  to  a  leukemic  that  it 
was  excreted  as  such  and  as  methyl  xanthin  in  about  the  same 
amounts  as  in  a  healthy  person.  According  to  Rost's*  experiments 
the  dog  will  excrete  about  31.8  per  cent,  of  the  administered  theo- 
bromin ;  the  rabbit  28  per  cent,  and  man  20  per  cent.  Unlike 
caffein,  the  base  produces  diuresis  in  the  dog  and  hence  the  much 
greater  elimination  of  theobromin  (page  345). 

About  the  same  time  Albanesi  experimented  with  caffein  and  like- 
wise obtained  a  methyl  xanthin  which  was  assumed  to  be  hetero- 
xanthin,  but  was  eventually  shown  to  be  3-methyl  xanthin  (p.  395). 
Kriiger  and  Schmidt  fed  theobromin  to  dogs  and  found  that  they 
eliminated  some  unchanged  base,  chiefly  3-methyl  xanthin  and  some 
7-methyl  xanthin  or  heteroxanthin.  In  rabbits  the  results  were  quali- 
tatively alike,  but  differed  quantitatively.  In  addition  to  theobromin 
their  urine  contained  chiefly  7-methyl  xanthin  and  but  little  3-methyl 
xanthin.  Later  Kriiger  showed  that  caffein  splits  up  in  the  dog  into 
theobromin  and  other  cleavage  products  (p.  406).) 

Caffein  or  1.3.7-tri-methyl  xanthin  (p.  340),  Q^^^p^,  is  the 
active  principle  of  tea  and  coffee  and  while  it  is  not  a  product  of 
tissue  metabolism  in  the  animal  body  it  is  of  interest  because  of  its 
relation  to  xanthin  and  its  derivatives  as  well  as  to  uric  acid.  Of  the 
ordinary  purin  bases  found  in  human  urine  only  adenin,  hypoxan- 
thin  and  xanthin  are  cleavage  products  of  the  nucleins  of  the  tis- 
sues. 1-  and  7-methyl  xanthins  (heteroxanthin)  and  1-7-di-methyl 
xanthin  (paraxanthin)  are  undeniably  derived  from  the  caffein  taken 

'  Berichte,  30,  1839  ;  31,  1980  ;  32,  470. 

^Benehte,  33,  3050. 

'^Arehiv.  Path.  u.  Pharm.,  36,  45,  133  ;  37,  385  ;  Berichte,  28,  1113, 

^  Archiv.  Path.  u.  Pharm.,  36,  56. 


406  CHEMISTRY  OF  THE  LEUCOMAINS. 

into  the  body.  Furthermore  3-methyl  xanthin  and  the  other  two 
di-methyl  xanthins,  theophyllin  and  theobromin,  appear  in  the  urine 
after  caffein  administration  and  are  therefore  likewise  cleavage  prod- 
ucts of  this  base. 

To  Bondzynski  and  Gottlieb  and  to  Albanesi  is  due  the  credit  of 
having  demonstrated  that  caffein  and  theobromin  were  demethylated 
within  the  animal  body.  The  former  worked  chiefly  with  theobromin 
and  showed  that  this  base  was  changed  in  dogs,  rabbits  and  in  man 
to  heteroxanthin.  Kriiger  and  Schmidt  eventually  showed  that  in 
addition  to  heteroxanthin  also  3-methyl  xanthin  was  formed. 

Albanesi  on  feeding  caffein  to  dogs  obtained  what  he  first  believed 
to  be  heteroxanthin  but  which  on  further  study  by  himself  and 
by  Kriiger  was  shown  to  be  3-methyl  xanthin.  The  investigations 
of  the  latter  are  the  most  exhaustive.  They  show  that  when  caffein 
is  given  to  the  dog  in  small  quantities  it  is  excreted  as  3-methyl 
xanthin.  In  large  doses,  however,  the  demethylation  is  not  so  com- 
plete and  hence  the  several  intermediate  products  appear.  Some 
caffein  passes  through  unchanged.  Each  of  the  three  methyl 
groups  are  attacked  in  the  organism  and  as  a  result  the  three  di- 
methyl xanthins,  theophyllin,  theobromin  and  paraxanthin,  appear  in 
the  urine.  The  first  mentioned  predominates  and  the  other  two  are 
present  in  about  equal  amounts.  3-methyl  xanthin  apparently  is  the 
only  mono-methyl  derivative  present.  The  demethylation  is  un- 
questionably different  in  different  animals  as  can  be  seen  from  ex- 
periments with  theobromin  (p.  405),  and  with  paraxanthin  (p.  403). 
Albanesi's  statement  that  caffein  in  rabbits  was  changed  to  xanthin, 
and  in  man  to  di-methyl  xanthin  (theophyllin  ?)  has  been  questioned 
by  Kriiger.  While  in  dogs  the  cleavage  of  caffein  yields  chiefly 
theophyllin  and  3-methyl  xanthin,  it  would  seem  that  in  man  para- 
xanthin and  1-  and  7-methyl  xanthins  are  the  more  resistant. 

It  seems  to  be  well  established  that  caffein  does  not  increase  the 
amount  of  uric  acid  in  the  urine.  As  shown  above,  however,  it  does 
materially  increase  the  amount  of  the  purin  bases.  Kriiger  and 
Schmid  have  shown  that  the  increase  of  purin  bases  is  not  propor- 
tional to  the  amount  of  caffein  administered.  Thus,  when  only 
0.05  g.  of  caffein  was  given  33  per  cent,  of  its  nitrogen  appeared  as 
purin  bases.  With  0.2  g.  of  caffein  only  19  per  cent,  of  the  nitro- 
gen was  found  in  this  condition.  The  ratio  of  the  nitrogen  of  uric 
acid  to  that  of  the  purin  bases  is  13.5  :  1  or  12.7  : 1.  After  caffein 
administration  it  falls  to  9.9  : 1  or  8.35  : 1. 

The  results  with  theobromin  were  in  some  respects  very  different. 
Like  caffein  it  does  not  increase  the  amount  of  uric  acid  but  it  has 
a  greater  influence  upon  the  purin  bases — the  ratio  being  2.6:1. 
In  other  words  47  per  cent,  of  the  theobromin  nitrogen  appeared  in 
the  form  of  purin  bases. 

As  yet  there  is  no  direct  evidence  going  to  show  that  xanthin  is 


SEPARATION  OF  THE  PURIN  BASES.  407 

formed  during  the  demethylation  of  caffein  and  of  the  other  higher 
homologues.  The  possibility  of  this  taking  place  must  be  conceded. 
On  the  other  hand  it  is  evident  that  the  purin  group  undergoes  to  a 
large  extent  complete  cleavage.  These  simpler  products  are  as  yet 
unknown  but  it  is  easy  to  see  that  pyrimidin  derivatives  and  even 
creatinin  may  originate  during  the  cleavage  of  purin  bodies.  Wiener's 
observations  on  uric  acid  show  that  this  purin  body  is  not  only  a  nuclein 
cleavage  product  but  that  it  may  also  be  a  synthetic  one.  Further- 
more he  has  shown  that  uric  acid  is  not  only  being  made  but  is  also 
being  constantly  destroyed  within  the  body.  This  process  of  for- 
mation and  of  decomposition  results  in  the  normal  minimum  excre- 
tion of  uric  acid  (see  page  344).  If,  however,  the  latter  change  is 
diminished  or  inhibited  the  amount  of  uric  acid  becomes  apparently 
increased  and  may  lead  to  disease  conditions  such  as  rheumatism. 
The  same  view  may  be  extended  to  the  purin  bases  and  the  rela- 
tively large  elimination  of  these  bodies  in  leukemia  may  be  due  not 
only  to  increased  formation  but  also  to  decreased  destruction. 

For  the  pharmacological  action  of  caffein  and  other  purins  see 
page  345. 

Caffein  was  discovered  in  1820  by  Runge.  It  can  be  readily 
prepared  from  xanthin  or  from  either  of  the  mono-methyl  or  di- 
methyl derivatives.  It  has  been  synthesized  from  the  mono-,  di-, 
tri-,  and  tetra-methyl  uric  acids.  In  other  words  it  can  be  prepared 
from  uric  acid  direct  (Fischer).  It  has  also  been  synthesized  from 
di-methyl  urea  and  cyanacetic  acid  (Traube). 

Separation  of  the  Purin  Bases. 

The  detection  and  separation  of  the  purin  bases  in  urine  offers 
considerable  difficulty  which  will  be  readily  understood  when  it  is 
remembered  that  as  many  as  fourteen  of  these  bases  have  been  found 
in  that  secretion.  Most  of  these,  as  indicated  elsewhere,  do  not 
represent  tissue  metabolism  proper  but  rather  cleavage  products  of 
the  purins  contained  in  the  food  and  drink. 

Neubauer's  method,  which  has  been  employed  in  some  modifica- 
tion or  other  for  the  isolation  of  purin  bases,  depends  upon  the  fact 
that  they  are  precipitated  from  an  ammoniacal  solution  by  an  am- 
moniacal  silver  solution.  This  reaction,  as  Kriiger  has  pointed  out, 
is  given  by  all  xanthin  bases  which  contain  an  imido  group  capable 
of  substitution.  It  is  not  given  by  caffein,  the  fully  methylated 
xanthin,  or  by  dimethyl  hypoxanthin.  The  further  separation  of 
the  bases  is  accomplished  by  dissolving  the  silver  precipitate, 
together  with  a  little  urea,  to  destroy  any  nitrous  acid,  in  boiling 
nitric  acid  of  1.1  specific  gravity.  The  hot  solution  is  filtered,  and 
from  the  filtrate,  on  cooling,  the  "  hypoxanthin  fraction  "  of  Salomon 
— hypoxanthin,  guanin,  carnin,  adenin,  and  episarkin,  crystallize 
as    the  corresponding  silver  nitrate   compounds.     That  portion  of 


408  CHEMISTRY  OF  THE  LEUCOMAINS. 

silver  salts  remaining  in  solution  in  the  nitric  acid,  the  "  xanthin 
fraction/'  consists  of  xanthin,  paraxanthin,  and  hetero xanthin. 

The  urine,  acidulated  with  hydrochloric  acid,  is  precipitated  with 
phosphotungstic  acid ;  the  precipitate  is  decomposed  by  warming 
with  baryta,  filtered,  and  the  filtrate  is  freed  from  barium  by  the 
cautious  addition  of  sulphuric  acid.  The  solution  is  then  made  alka- 
line with  ammonium  hydrate,  any  traces  of  phosphates  that  appear 
are  filtered  off.  Or,  the  urine  may  be  rendered  directly  alkaline 
with  ammonium  hydrate  and  after  standing  24  hours  the  precipi- 
tated phosphates  removed  by  filtration.  The  filtrate  finally  is  pre- 
cipitated by  the  addition  of  ammoniacal  silver  nitrate  (0.5  g.  per 
liter).  The  precipitate  which  forms  consists  of  the  silver  compounds 
of  the  xanthin  bodies,  and  is  purified  by  dissolving  in  boiling  nitric 
acid  of  1.1  sp.  g.  The  filtrate,  on  cooling,  yields  a  precipitate  of 
the  silver  nitrate  compounds  of  adenin,  hypoxanthin,  and  guanin. 
The  xanthin  compound  remains  in  solution.  The  silver  salts  of  the 
three  bases  mentioned  are  decomposed  with  dilute  hydrochloric,  or 
with  hydrogen  or  sodium  or  ammonium  sulphid,  and  the  acid  solu- 
tion of  the  three  bases  is  then  heated  on  the  water-bath  with  excess 
of  ammonia.  Guanin  is  thus  thrown  out  of  solution.  In  the  am- 
moniacal filtrate  the  adenin  is  separated  from  the  hypoxanthin  and 
estimated  by  picric  acid  (see  p.  411).  The  hypoxanthin  is  esti- 
mated as  hypoxanthin  silver  picrate.  Instead  of  the  separation  of 
guanin  from  the  other  bases  by  ammonia  Wulff's  metaphosphate 
method  may  be  employed  (p.  385).  The  adenin  and  hypoxanthin 
are  then  precipitated  from  the  filtrate  by  ammoniacal  silver  solution. 
The  silver  salts  decomposed  with  hydrochloric  acid,  and  in  the 
filtrate  the  adenin  can  be  estimated  as  the  picrate ;  the  hypoxanthin 
as  the  hypoxanthin  silver  picrate. 

A  second  method  for  the  extraction  of  the  xanthin  bases  is  based 
upon  Drechsel's  reaction,  namely,  that  they  are  precipitated  by  a  cop- 
per solution  in  the  presence  of  a  reducing  agent  (see  page  353).  The 
precipitation  is  so  complete  that  the  filtrate  does  not  react  with  am- 
moniacal silver  nitrate.  The  reliability  of  the  process  has  been 
quantitatively  tested  by  Balke,  who  found  the  yield  of  xanthin  bases 
from  meat  extract  to  be  slightly  greater  by  the  copper  method  over 
Neubauer's  silver  nitrate  process.  In  addition  to  cheapness  the 
method  possesses  an  advantage  in  the  less  bulky  precipitates. 

As  reagents  Balke  employed  Fehling's  solution  and  hydroxylarain 
hydrochlorid,  whereas  Kriiger  made  use  of  copper  sulphate  (13  per 
cent.)  and  sodium  bisulphite  (1-2).  The  latter  solutions  are  ob- 
viously preferable. 

The  urine  or  other  liquid  to  be  examined  is  first  freed  from  any 
albumin  that  may  be  present.  It  is  then  boiled  and  the  copper  and 
bisulphite  solutions  are  added.  Barium  chlorid  may  be  added  to 
facilitate  the  settling  of  the  precipitate,  which  after  standing  several 


SEPARATION  OF  THE  PURIN  BASES.  409 

hours  is  filtered  off  and  repeatedly  washed  with  warm  water  (60°). 
The  precipitate  is  then  suspended  in  acidulated  water  and  decom- 
posed with  hydrogen  sulphid.  To  destroy  the  uric  acid  which  may 
be  present  the  solution  is  acidulated  with  acetic  acid  and  boiled  with 
manganese  dioxid,  after  which  the  dissolved  manganese  is  precipi- 
tated by  boiling  with  ammonium  carbonate  and  hydrate.  The  purin 
bases  may  now  be  reprecipitated  with  copper  sulphate  and  bisulphite 
solutions. 

All  the  xanthin  compounds  containing  an  imido  group  capable  of 
substitution,  with  the  exception  of  theobromin,  are  precipitated. 
Caffein  and  dimethyl  hypoxanthin  contain  no  imido  group,  and  are 
therefore  not  precipitated.  Neither  are  urea,  allantoin,  amido  acids, 
pepton,  albumose,  creatin  and  creatinin  thrown  down.  The  more 
imido  groups  present  in  a  compound  the  less  soluble  is  the  precipitate. 

Kriiger  and  Wulff  in  1894  applied  the  copper  method  for  the 
determination  of  the  relative  amounts  of  purin  bases  and  uric  acid 
in  urines  (see  Novy's  Physiological  Chemistry).  The  ratio  of  the 
nitrogen  of  the  former  to  that  of  the  latter  is  of  little  value  when  it 
is  remembered  that  the  greater  part  of  the  purin  bases  in  the  urine 
are  derived  from  the  caffein  and  similar  bodies  in  the  food  and  have 
nothing  to  do  with  cell  metabolism. 

The  Neubauer  method  as  ordinarily  employed  does  not  yield  as 
satisfactory  results  as  may  be  expected.  As  pointed  out  by  Kriiger 
and  Salomon  the  use  of  nitric  acid  would  cause  an  oxidation  of 
carnin  to  hypoxanthin.  Moreover,  any  nitrous  acid  present  would 
convert  more  or  less  completely  the  guanin  into  xanthin  and  the 
adenin  into  hypoxanthin.  This  change  as  Kriiger  has  shown  is  not 
prevented  by  the  addition  of  even  a  large  excess  of  urea.  They  thus 
explain  their  non-detection  of  carnin  and  guanin  in  the  urine.  A 
more  serious  objection  was  pointed  out  by  them  in  connection  with 
the  separation  of  the  purin  bases  into  the  two  fractions  by  means  of 
boiling  nitric  acid.  The  supposition  that  the  silver  salts  of  the 
xanthin  fraction  are  easily  soluble  in  the  nitric  acid  is  not  correct. 
Thus,  the  silver  salt  of  heteroxanthin  requires  2,820  parts  of  nitric 
acid  (1.1  sp.  gr.)  for  solution.  It  follows  therefore  that  the  hypo- 
xanthin fraction  as  ordinarily  obtained  always  contains  considerable 
amounts  of  the  xanthin  fraction. 

In  the  course  of  their  exhaustive  investigation  upon  the  urinary 
bases  Kriiger  and  Salomon  ^  recognized  these  defects  and  devised  the 
following  method  of  separation : 

The  purin  bodies  may  be  precipitated  either  by  means  of  ammon- 
iacal  silver  solution  or  by  means  of  copper  sulphate  and  sodium 
bisulphite  as  already  described.  In  the  former  case  the  washed  pre- 
cipitate is  transferred  to  a  round  bottom  flask  and  decomposed  on  a 

^Zdts.  physiol.  Chein,,  26,  373  (1898). 


410  CHEMISTRY  OF  THE  LEUCOMAINS. 

water-bath  by  cautious  addition  of  dilute  hydrochloric  acid  till  the 
original  voluminous  precipitate  is  replaced  by  that  of  silver  chlorid. 
The  liquid  is  then  heated  over  a  flame  and  hydrochloric  acid,  equal 
to  that  already  used,  is  added.  The  solution  is  filtered  while  hot 
and  the  precipitate,  which  contains  most  of  the  uric  acid,  is  thoroughly 
washed  with  very  dilute  hydrochloric  acid. 

When  the  copper  method  is  used  the  precipitate,  after  thorough 
washing  with  boiling  water,  is  transferred  to  a  flask,  then  warmed 
and  rendered  alkaline  with  ammonium  hydrate.  The  liquid  is  then 
strongly  acidulated  with  hydrochloric  acid  and  the  copper  is  pre- 
cipitated by  means  of  hydrogen  sulphid.  The  precipitate  is  filtered 
while  hot  and  freely  washed.  It  contains  the  greater  part  of  the 
uric  acid. 

The  acid  filtrate  obtained  by  either  method  is  concentrated  on 
the  water-bath,  preferably  at  a  low  temperature  and  with  constant 
stirring.  To  remove  the  acid  from  the  syrupy  residue  this  is  twice 
taken  up  with  water  and  reevaporated.  This  operation  is  repeated 
with  96  per  cent,  alcohol  till  the  acid  is  expelled  and  the  residue  be- 
comes granular.  This  is  then  digested  for  several  hours  at  40°  with 
distilled  water ;  the  insoluble  part  is  filtered  off  and  washed  with  water, 
then  with  alcohol  and  with  ether.  The  aqueous  filtrate,  evaporated 
and  the  residue  treated  as  before,  may  yield  a  slight  insoluble  por- 
tion which  is  to  be  combined  with  the  main  quantity. 

The  insoluble  residue  contains  xanthin,  heteroxanthin,  and 
1 -methyl  xanthin.  The  aqueous  filtrate  contains  adenin,  hypoxan- 
thin,  paraxanthin  and  epiguanin  with  traces  of  heteroxanthin  and 
1-methyl  xanthin.  In  other  words  xanthin  and  hypoxanthin  frac- 
tions result  the  same  as  in  Neubauer's  method  with  the  difference 
that  paraxanthin  appears  in  the  latter. 

1.  In  order  to  separate  the  three  bases  present  in  the  xanthin 
fraction  the  insoluble  residue  is  dissolved  in  fifteen  parts  of  hot  3.3 
per  cent,  sodium  hydrate  (HCl  free).  Within  24  hours  the  sodium 
salt  of  heteroxanthin  separates  out  pure  and  almost  quantitatively. 

This  is  filtered  off  and  the  filtrate  is  divided  into  portions  of  60 
c.c.  and  warmed  to  60°.  Each  portion  is  then  poured  slowly  and 
with  constant  stirring  into  a  cold  mixture  of  20  c.c.  of  concentrated 
nitric  acid  and  20  c.c.  of  water.  This  dilute  acid  should  be  boiled 
before  being  used.  The  uric  acid  remaining  in  solution  is  thus  de- 
stroyed and  the  solution  on  standing  a  few  hours  in  the  cold  yields 
a  precipitate  of  xanthin  nitrate.  This,  if  pure,  is  a  heavy  crystal- 
line powder  consisting  of  masses  of  plates.  If  impure,  the  air- 
dried  precipitate  should  be  divided  into  portions  of  3  g.,  each  of 
which  is  then  dissolved  in  warm  sodium  hydrate  and  the  solution 
diluted  to  60  c.c.  is  treated  as  before.  To  obtain  free  xanthin  the 
nitrate  is  dissolved  in  ammonia  and  from  the  solution  when  evapo- 
rated xanthin  separates  out  in  amorphous  crusts. 


SEPARATION  OF  THE  PURTN  BASES.  41 1 

The  filtrate  from  the  xanthin  nitrate  saturated  with  ammonia  and 
concentrated  yields  a  satin-like  mass  consisting  of  microscopic  plates 
of  \~methyl  xanthin.  The  remainder  in  solution  can  be  precipitated 
with  ammoniacal  silver  solution  or  with  copper  sulphate. 

2.  The  filtrate  from  the  xanthin  fraction  on  addition  of  a  slight 
excess  of  ammonia  yields  at  once  a  precipitate  of  glistening  prisms 
—  epiguanin.  This  is  filtered  off  and  the  ammonia  is  expelled  from 
the  filtrate  by  heating.  The  solution  which  should  not  be  too  con- 
centrated is  then  treated  in  the  cold  with  a  1.1  per  cent,  solution  of 
picric  acid  to  a  slight  excess.  The  adenin  picrate  is  at  once  removed 
by  means  of  a  suction  pump. 

The  filtrate  from  the  adenin  is  acidulated  with  sulphuric  acid  and 
extracted  with  benzol  or  toluol  to  remove  picric  acid.  After  which 
the  remaining  bases  are  precipitated  with  ammoniacal  silver  solution 
or  with  copper  sulphate  and  bisulphite.  The  precipitate  is  decom- 
posed with  hydrogen  sulphid  after  which  the  aqueous  filtrate  is 
evaporated  to  dryness.  Portions  of  3  g.  each,  of  the  dry  residue, 
are  dissolved  in  100  c.c.  of  hot  dilute  nitric  acid  (90  c.c.  H^O 
-j-  10  c.c.  cone.  HNO3).  On  cooling  pure  hypoxanthin  nitrate  sep- 
arates out. 

The  filtrate  from  this  deposit  contains  small  amounts  of  hypoxan- 
thin, heteroxanthin,  and  1-methyl  xanthin  besides  paraxanthin.  To 
separate  these  bases  the  entire  method  is  repeated  from  the  begin- 
ning. The  above  filtrate  is  precipitated  with  ammoniacal  silver  so- 
lution or  with  copper  sulphate  and  sodium  bisulphite.  The  hydro- 
chloric acid  filtrate,  obtained  by  decomposing  the  precipitate  in  the 
manner  previously  described,  is  evaporated  and  the  residue  is  ex- 
tracted as  before  with  as  little  cold  water  as  possible.  The  insoluble 
portion  contains  heteroxanthin  and  1-methyl  xanthin,  and  these  are 
separated  by  means  of  a  3.3  per  cent,  solution  of  sodium  hydrate. 
The  filtrate  contains  hypoxanthin  and  paraxanthin.  In  order  to  re- 
move the  hydrochloric  acid  these  bases  are  again  precipitated  as 
silver  or  copper  compounds.  These  are  decomposed  and  the  dry 
residue  dissolved  in  a  little  hot  dilute  nitric  acid  (as  above)  yields  on 
cooling  hypoxanthin  nitrate.  The  filtrate  contains  paraxanthin 
which  can  be  obtained  now  as  the  sodium  salt  or  as  the  free  base. 

Guanin,  if  present,  is  to  be  expected  in  both  fractions.  The  xan- 
thin fraction,  treated  with  ammonia,  would  yield  the  insoluble  guanin. 
From  the  hypoxanthin  fraction  guanin  and  epiguanin  would  be  pre- 
cipitated together  by  ammonia.  It  can  be  separated  from  the  latter 
by  treatment  with  hot  water  or  hot  dilute  ammonia. 

Inasmuch  as  caffein  and  theobromin  are  not  precipitated  by  am- 
moniacal silver  solution  or  by  the  copper  sulphate  method  it  is  neces- 
sary in  order  to  detect  these  bases  to  resort  to  precipitation  with 
phosphotungstic  acid.  For  this  purpose  the  urine,  acidulated  with 
sulphuric  acid,  is  treated  with  this  reagent.     The  precipitate  is  de- 


412  CHEMISTRY  OF  THE  LEUCOMAINS. 

composed  in  the  cold  with  baryta  water,  treated  with  carbonic  acid 
and  finally  the  liquid  is  warmed  on  the  water-bath  and  filtered  hot. 
The  filtrate  is  then  precipitated  with  copper  sulphate  and  sodium 
bisulphite  to  remove  the  ordinary  purin  bases.  The  filtrate  from  the 
copper  precipitate  is  treated  with  hydrogen  sulphid,  the  resulting 
clear  solution  is  evaporated  and  the  residue  extracted  with  chloro- 
form. This  takes  up  the  caffein  and  theobromin.  To  separate 
these  the  chloroform  solution  is  evaporated  and  the  residue  is  taken 
up  with  water,  and  the  solution  treated  with  silver  nitrate  and  a 
slight  excess  of  ammonia.  The  ammonia  is  then  expelled  by  boil- 
ing and  the  solution  on  cooling  yields  a  deposit  of  the  theobromin 
silver  compound.  The  filtrate  from  this  deposit  is  acidulated  with 
hydrochloric  acid  and  extracted  with  chloroform,  which  on  evapo- 
ration yields  caffein. 

The  3-methyl  xanthin  is  separated  from  the  purin  bases  as  the  in- 
soluble barium  salt.  Theophyllin  is  separated  from  paraxanthin  by 
recrystallizLng  the  silver  nitrate  compounds  from  nitric  acid  (Kriiger).^ 

Separation  of  uric  acid  from  xanthin  bases. 

In  the  precipitation  of  uric  acid  by  the  silver  method  the  xanthin 
bases  if  present  are  likewise  thrown  down.  However,  as  adenin  and 
hypoxanthin  are  readily  soluble  in  acids  and  alkalis,  they  are  easily 
separated  after  decomposition  of  the  silver  precipitate.  Xanthin 
and  guanin  are  less  soluble,  and  hence  may  render  the  uric  acid 
impure  especially  when  this  is  isolated  from  animal  organs.  Wulff 
detected  the  presence  of  xanthin  in  a  uric  acid  precipitate  by  destroy- 
ing the  uric  acid  with  hot  dilute  nitric  acid — the  xanthin  remaining 
unchanged.  The  substance  is  heated  on  the  water-bath  with  dilute 
nitric  acid  (100  parts  water  and  5  parts  nitric  acid,  1.14  sp.  g.),  and 
after  gas  ceases  to  be  given  off  it  is  boiled  for  a  short  time,  rendered 
ammoniacal,  and  if  xanthin  is  present  it  gives  a  precipitate  with 
silver  nitrate.  The  precipitate  can  be  collected  on  a  weighed  filter, 
dried  at  120°,  and  weighed  as  C^H^N^Og-AgjO  ;  or  from  the  weight 
of  silver  after  ignition  the  amount  of  xanthin  may  be  calculated.  If 
the  amount  of  xanthin  is  large,  it  can  be  weighed  directly.  For  this 
purpose  the  solution  is  rendered  slightly  ammoniacal,  after  the 
oxidation  with  nitric  acid,  and  then  is  treated  with  acetic  acid  and 
an  equal  volume  of  alcohol.  After  twelve  hours  filter,  wash  with 
dilute  alcohol,  dry  at  110°,  and  weigh. 

The  destruction  of  uric  acid  by  nitric  acid  and  by  manganese 
dioxid  has  been  already  referred  to.  Horbaczewski's  method  for  the 
separation  of  uric  acid  from  xanthin  is  as  follows  :  The  mixture  of 
the  two  substances  is  dissolved  with  aid  of  gentle  heat  in  a  platinum 
dish  in  concentrated  sulphuric  acid  (2  c.c.  for  0.1  g.  substance). 
>  Benchte,  32,  2681,  2823,  3336. 


CABNIN.  413 

The  solution  is  diluted  with  four  parts  of  water,  stirred  thoroughly, 
and  set  aside  for  3-6  hours.  The  uric  acid  is  thus  precipitated,  and 
is  then  collected  on  a  small  filter,  washed  with  water,  acidulated  with 
sulphuric  acid,  then  with  pure  water.  Then  it  is  transferred  to  the 
dish  in  which  it  was  precipitated,  dissolved  in  a  little  pure  sodium 
hydrate  (e  natrio),  strongly  acidulated  with  hydrochloric  acid,  and 
evaporated  to  a  few  c.c.  After  standing  one  hour  it  is  filtered 
through  Ludwig's  glass-wool  filter,  washed  with  HCl-water,  then 
with  water,  finally  with  alcohol  and  ether,  dried  at  110°,  and 
weighed.  The  filtrate  and  wash-water  are  combined,  and  a  correc- 
tion is  made  for  the  solubility  (1  :  16,000)  of  uric  acid.  This  correc- 
tion is  added  to  the  weighed  amount.  The  results  obtained  by  this 
method  are  excellent.  The  separation  from  guanin  and  xanthin  is 
complete. 

Carnin,  C^HgN^Og,  was  isolated  in  1871  from  American  meat 
extract  by  Weidel,  but  it  has  not  been  obtained  from  muscle  tissue 
itself.  Balke,  however,  has  isolated  it  from  fresh  horse-meat  extract. 
It  has  also  been  obtained  from  yeast  liquors  by  Schiitzenberger,  and 
Pouchet  claims  to  have  isolated  it  from  urine,  but  has  not  offered 
sufficient  evidence  on  this  point.  Salomon  (1893)  obtained  a  body 
resembling  carnin  from  leuksemic  urine. 

It  can  be  separated  from  the  meat  extract  by  the  following  method 
originally  employed  by  Weidel :  The  extract  is  dissolved  in  six  or 
seven  parts  of  warm  water,  then  concentrated  baryta  water  is  added, 
avoiding,  however,  an  excess.  The  filtrate  is  precipitated  by  basic 
lead  acetate.  The  precipitate  is  collected,  thoroughly  washed  and 
pressed,  and  finally  it  is  repeatedly  extracted  with  a  large  quantity 
of  boiling  water.  The  carnin  lead  salt  is  thus  dissolved  out ;  the 
filtrate,  after  removal  of  the  lead  by  hydrogen  sulphid,  is  evaporated 
to  a  small  volume.  The  concentrated  solution  thus  obtained  is 
treated  with  silver  nitrate,  which  gives  a  precipitate  of  silver  chlorid 
and  of  the  silver  salt  of  carnin.  By  treatment  with  ammonium  hy- 
drate the  silver  chlorid  can  be  completely  removed  from  the  precipi- 
tate, whereas  the  silver  compound  of  carnin  is  insoluble  in  that 
reagent.  To  obtain  pure  carnin  the  silver  salt  is  decomposed  with 
hydrogen  sulphid,  and  the  filtrate,  after  purification  by  bone-black, 
is  evaporated  to  crystallization.  According  to  Weidel,  carnin  forms 
about  1  per  cent,  of  the  meat  extract.  Kemmerich  (1893)  has  found 
only  one-quarter  of  1  per  cent.,  or  even  less.  The  amount,  there- 
fore, varies  considerably,  and  may  be  very  small  in  the  fresh  extract. 

Carnin  forms  white  crystalline  masses  which  on  drying  become 
loose  and  chalk-like.  It  is  very  difficultly  soluble  in  cold  water, 
easily  and  completely  in  boiling  water,  and  recrystallizes  on  cooling. 
It  is  insoluble  in  alcohol  and  ether.  The  taste  is  decidedly  bitter, 
and  the  reaction  is  neutral.     The  base  is  not  precipitated  by  neutral 


414  CHEMISTRY  OF  THE  LEUCOMAINS. 

lead  acetate,  but  is  precipitated  by  the  basic  salt  as  a  flocculent  white 
precipitate  soluble  in  boiling  water.  On  heating  carnin  decomposes 
and  takes  fire,  and  at  the  same  time  gives  off  a  peculiar  odor.  It 
crystallizes  with  one  molecule  of  water,  which  it  loses  at  100°-110**. 

The  hydrochloride  C^HgN^Og.HCl,  forms  pretty  prisms  (Kem- 
merich),  and  decomposes  on  heating  with  concentrated  hydrochloric 
acid. 

The  platinochlorid,  C^HgN^Og-HCLPtCl^ ,  forms  a  fine,  sandy, 
golden-yellow  powder. 

With  silver  nitrate  carnin  unites  to  form  a  white  flocculent  pre- 
cipitate, insoluble  in  nitric  acid  or  in  ammonia  hydrate.  Its  formula 
corresponds  to  2(C.HyAgNP3)  +  AgNOg . 

With  copper  solution  and  reducing  agent  it  gives  a  yellow  pre- 
cipitate which  on  washing  becomes  grass-green  (Balke). 

Carnin  is  not  affected  by  prolonged  boiling  with  concentrated 
barium  hydrate.  Bromin  water  decomposes  it  with  the  evolution  of 
gas  and  the  formation  of  hypoxanthin.  This  change  takes  place 
according  to  the  following  equation  : 

CyHgN.Og  +  2Br  =  C^H.Np.HBr  +  CHgBr  -f  CO^ . 

A  similar  decomposition  into  hypoxanthin  is  brought  about  by  the 
action  of  nitric  acid,  though  in  this  case  oxalic  acid  and  a  yellow 
body  are  formed.  Because  of  this  decomposition  carnin  cannot  be 
expected  when  Neubauer's  method  is  used.  When  carnin  is  evapo- 
rated with  chlorin  water  containing  a  little  nitric  acid,  the  residue, 
on  contact  with  ammonia,  gives  a  rose-red  color  (murexid  test).  This 
is  due,  according  to  Weidel,  to  the  formation  of  hypoxanthin,  but  it 
has  since  been  shown  that  the  latter  base  does  not  give  this  reaction 
which,  however,  is  given  by  xanthin  and  its  derivatives. 

The  constitution  of  carnin  is  not  known.  It  is  undoubtedly  an 
ester  of  hypoxanthin.  The  above  decompositions  may  be  taken  to 
indicate  the  presence  of  an  acetic  group  in  position  7  ;  otherwise  it 
must  be  looked  upon  as  a  methyl  carboxyl  derivative  of  hypoxanthin. 

The  physiological  action  of  carnin  has  been  examined  somewhat 
by  Briicke,  and  according  to  him  the  base  is  not  very  poisonous. 
The  only  effect  observed,  when  taken  internally,  was  a  fluctuation 
in  the  rate  of  the  heart-beat,  though  even  this  was  by  no  means 
definite  in  its  nature. 

Carnosin,  Cgllj^NPg,  is  a  new  base  isolated  in  1900  by  Gule- 
witsch  and  Amiradzibi^  from  Liebig's  meat  extract.  It  has  not  as 
yet  been  studied  very  thoroughly  and  nothing  definite  can  be  said  as 
to  its  structure.  The  silver  compounds  show,  however,  a  striking 
resemblance  to  arginin.     It  may  be   looked   upon  as  a  di-methyl 

'  Berichte,  33,  1902. 


PYRIMIDIN  GROUP.  415 

carnin  in  which  case  it  would  contain  less  hydrogen  than  given 
above. 

The  free  base  is  very  easily  soluble  in  water  and  has  a  strong 
alkaline  reaction.  It  forms  minute  needle-shaped  crystals  and  melts 
with  decomposition  at  239°. 

The  nitrate  on  evaporation  yields  radiating  crystals  and  is  also 
precipitated  in  stellate  masses  of  needles  by  the  addition  of  alcohol 
to  a  hot  aqueous  solution  of  the  salt.  It  is  dextro-rotatory  and  melts 
with  decomposition  at  211°. 

On  boiling  with  copper  carbonate  it  yields  a  copper  compound 
(CgH^^N^Og.CuO)  which  is  difficultly  soluble  in  cold,  rather  easily 
soluble  in  hot  water  from  which  it  crystallizes  in  characteristic  six- 
sided  plates.     It  decomposes  without  melting  at  220°. 

Cytosin,  CjjHgpNjgO^  +  SHjO,  was  obtained  by  Kossel  and  Neu- 
mann by  the  decomposition  of  adenylic  acid  (from  thymus  glands  ) 
by  heating  with  20  percent,  sulphuric  acid  in  a  sealed  tube  at  150°  ; 
also  by  the  action  of  water  at  170°.  The  yield  is  about  2  per  cent. 
It  forms  rectangular  plates,  often  with  blunted  corners.  On  slow 
separation  the  crystals  may  attain  a  length  of  a  centimeter.  The 
water  of  crystallization  is  expelled  at  100°.  It  is  easily  soluble  in 
hot  water,  from  which  it  separates  on  cooling ;  difficultly  soluble  in 
alcohol ;  insoluble  in  ether. 

It  forms  well  crystallized  salts.  Thus,  the  sulphate  forms  needles  ; 
the  chlorid  is  easily  soluble  and  appears  in  prisms.  The  nitrate, 
platinochlorid,  and  aurochlorid  likewise  crystallize  easily.  A  brick- 
red  precipitate  forms  in  even  very  dilute  acidulated  solution  by  the 
addition  of  potassium  bismuth  iodid.  Silver  nitrate  produces  a  pre- 
cipitate which  is  increased  by  addition  of  a  little  ammonia,  but  is 
dissolved  gradually  by  an  excess,  especially  on  warming,  but  it  reap- 
pears in  crystalline  form  on  cooling. 

The  picrate,  C2iH3,N„0,.2CgH3N307,  is  difficultly  soluble  and 
crystallizes  in  yellow  needles. 

It  is  evident  that  cytosin  is  still  subject  to  investigation.  In  some 
respects  it  suggests  a  relationship  to  the  pyrimidin  bodies.  At  all 
events  the  formula  as  given  is  more  complex  than  might  be  expected 
by  comparison  with  the  other  well  studied  products  of  nucleins. 

PYRIMIDIN  GROUP. 

Two  substances  belonging  to  this  group  have  been  isolated  from 
nucleinic  acids  by  hydrolysis.  Although  they  are  neither  basic  nor 
acid  in  character,  yet  their  relation  to  the  purin  bodies  and  hence 
to  nuclein  is  such  as  to  render  their  consideration  necessary.  They 
have  been  regarded  as  antecedents  of  the  purin  bodies,  rather  than 
as   decomposition  products  of  these  substances.     Nevertheless,  it 


416  CHEMISTRY  OF  THE  LEUCOMAINS. 

would  seem  as  if  the  latter  view  was  the  correct  one.  The  reason 
for  this  belief  is  twofold.  In  the  first  place  the  hydrolytic  process 
is  so  energetic  that  adenin  and  presumably  the  other  purin  bases  as 
well  are  completely  broken  up.  Again,  if  the  pyrimidin  bodies 
are  antecedents,  it  might  be  expected  that  on  feeding  to  animals 
purin  bodies  would  be  eliminated  in  the  urine.  Steudel  (1901),  how- 
ever, has  shown  that  a  number  of  synthetic  pyrimidin  bodies  can 
be  given  to  the  dog  with  the  result  that  some  of  these  are  destroyed 
while  others  are  eliminated  as  such,  and  at  no  time  are  purin  bodies 
formed.  Thymin,  a  natural  pyrimidin  body,  is  split  up  and  yields 
urea,  whereas  its  isomer  the  synthetic  methyl  uracil  is  excreted  un- 
changed. 

Steudel's  investigation  further  shows  that  the  di-  and  tri-amino 
derivatives  of  pyrimidin  are  highly  toxic,  while  the  others  are  not. 
These  poisonous  substances  yield  insoluble  compounds  which  are 
deposited  in  the  tubules  of  the  kidney.  It  is  noteworthy  that  the 
really  only  poisonous  natural  purin  body  is  adenin  which,  as  already 
shown,  is  6-amino  purin  (see  pages  341,  350).  It  would  seem  as 
if  the  introduction  of  the  amino  group  into  the  purin  or  pyrimidin 
nucleus  rendered  such  bodies  poisonous. 

Thymin. — In  1893,  Kossel  and  Neumann  showed  that  the  nu- 
cleinic  acid  from  the  thymus  gland  of  the  calf  on  boiling  with  dilute 
sulphuric  acid  yields  a  crystalline  body  (8  per  cent.)  which  they  des- 
ignated as  thymin.  At  first  they  ascribed  it  the  formula  CggHjgNgOg 
but  subsequent  investigations  showed  that  the  original  preparations 
were  not  pure  and  that  thymin  was  Q^^f)^ . 

Thymin  was  also  obtained  by  Kossel  and  Neumann  by  similar 
hydrolysis  of  the  nucleinic  acid  of  ox  spleen.  The  "  nucleosin " 
which  Miescher  isolated  from  a  nucleinic  acid  derived  from  the  sper- 
matozoa of  the  salmon  is  according  to  Kossel  identical  with  thymin. 

In  1896  Kossel  isolated  it  from  the  spermatozoa  of  the  sturgeon 
and  three  years  later  Gulewitsch  obtained  it  from  the  testicles  of  the 
herring.  Its  possible  presence  in  the  auto-digestion  of  the  pancreas 
has  been  indicated  by  Levene.  Kutscher  obtained  thymin  and  pos- 
sibly uracil  in  the  auto-digestion  of  thymus  glands.  Fischer  and 
Roeder^  in  1901  prepared  thymin  and  uracil  synthetically. 

Thymin  forms  four  or  six-sided  colorless  plates  resembling  those 
of  cholesterin  and  is  doubly  refractive.  Illustrations  of  these  are 
given  by  Gulewitsch  {Zeit.  physiol.  Chem.,  27,  295).  On  careful 
heating  it  can  be  sublimed  and,  as  Kossel  has  shown,  its  presence  in 
nucleinic  acid  can  thus  be  directly  demonstrated  by  heating  some  of 
the  acid  between  two  watch  glasses.  The  melting  point  is  above 
290°.  "When  heated  rapidly  it  sinters  at  318°  and  melts  at  about 
321°  with  evolution  of  gas  becoming  yellowish  (Fischer  and 
^Berichte,  34,  3751,  4129. 


PYRIMIDIN  GROUP.  417 

Roeder).  It  is  difficultly  soluble  in  cold,  easily  so  in  boiling  water, 
less  easily  in  alcohol  than  in  ether.  Although  it  is  neither  acid 
nor  basic  in  character  a  potassium  thymin  compound  can  be  obtained 
(Steudel),  with  mercuric  nitrate  it  yields  a  voluminous  precipitate. 
Mercuric  chlorid  gives  no  precipitate  but  does  form  one  if  the 
liquid  containing  the  mercury  salt  is  rendered  slightly  alkaline  with 
sodium  hydrate.  In  like  manner  silver  nitrate  does  not  yield  a  pre- 
cipitate but  the  subsequent  addition  ammonium  or  barium  hydrate 
produces  a  gelatinous  precipitate  which  redissolves  in  excess  of  the 
alkali. 

Thymin  decolors  bromin  water  and  eventually  forms  a  bromin 
derivative.  According  to  Jones  (1900)  this  change  takes  place 
according  to  the  equation  : 

CgHgNPj  +  2Br  +  Hp  =  C^H^N^OgBr  +  HBr. 

On  heating  with  phosphorus  oxychlorid  Steudel  and  Kossel  (1900), 
obtained  a  dichlorthymin  which  was  different  from  that  obtained  from 
the  isomer  methyl  uracil.  By  heating  potassium  thymin  with  methyl 
iodid  Steudel  obtained  a  dimethyl  thymin  which  also  differed  from 
the  isomeric  trimethyl  uracil  of  Behrend. 

Although  at  the  time  of  the  discovery  of  thymin  Kossel  suggested 
that  it  possibly  might  be  a  pyrimidin  body,  yet  positive  proof  of  this 
and  the  demonstration  of  its  constitution  was  not  supplied  until  re- 
cently. The  pyrimidin  character  was  demonstrated  by  Steudel  (1901), 
who  succeeded  in  obtaining  a  nitro-thymin  and  this  on  reduction  with 
tin  and  hydrochloric  acid  gave  a  body  which  with  chlorin  water  and 
ammonia  yielded  an  intense  murexid  reaction,  thus  indicating  the 
presence  of  the  alloxan  group.  This  was  further  corroborated  by  the 
oxidation  of  thymin  with  barium  permanganate,  urea  being  obtained. 
In  view  of  these  reactions  thymin  is  a  pyrimidin  body  isomeric  with 
methyl  uracil.  The  di-substitution  products  indicate  that  thymin  pos- 
sesses the  following  structural  formula : 

HN— CO  HN— CO  -tf~9^ 

OC     C— CH,  OC     CH  HC     CH 


HN— CH  HN— CH  N=CH 

Thymin.  Uracil.  Pvrimidiw. 

CjH„N,Oj.  C,H,N,0,.  C,H,N,. 

The  position  of  the  carbon  and  nitrogen  is  indicated  by  numbers 
in  the  same  way  as  in  the  case  of  purin  (p.  338).  Thymin  is  there- 
fore 5 -methyl  2-6-dioxypyrimidin.  Methyl  uracil,  the  isomer  syn- 
thesized by  Behrend  and  Roosen,  has  the  methyl  group  in  position 
4  ( =  6).  It  is  prepared  by  the  action  of  urea  on  acetacetic  ether. 
Gabriel  and  Colman  ^  changed  methyl  uracil  into  4  ( =  6)  methyl 
pyrimidin  and  eventually  into  pyrimidin.     From  the  4.5.6-metLyl 

^Berichte.  32,1537  (1899). 
27 


418  CHEMISTRY  OF  THE  LEUCOMAINS. 

diamido  pyrimidin  by  the  action  of  formic  acid  they  prepared  6- 
methyl  purin/  thus  effecting  the  conversion  of  a  pyrimidin  into  a 
purin  body.  Obviously  the  reverse  change  is  equally  possible,  in 
which  case  the  origin  of  the  pyrimidin  bodies  is  easily  accounted  for. 
Pyrimidin  has  also  been  synthesized  by  Emery.*  Several  amido  and 
methyl  derivatives  have  been  recently  described.^ 

From  the  above  constitution  of  pyrimidin  it  will  be  seen  that  it 
may  be  looked  upon  as  benzol,  in  which  the  nitrogen  occupies  the 
meta  position.  It  therefore  has  two  isomers.  Thus,  when  the 
nitrogen  is  in  the  ortho  position  pyridazin  results  ;  when  in  the  para 
position  pyrazin. 

Uracil. — From  yeast  nucleinic  acid  Kossel  and  Neumann  in  1893 
isolated  a  thymin-like  substance  but  the  amount  obtained  was  insuffi- 
cient for  identification.  Ascoli,  in  1901,  demonstrated  that  this  sub- 
stance was  not  thymin  but  the  next  lower  homologue,  C^H^NgOj , 
which  corresponds  to  the  hitherto  unknown  uracil.  A  similar,  if 
not  identical  body,  with  possibly  thymin,  is  formed  in  the  auto- 
digestion  of  the  pancreas  (Levene,  1901)  and  it  is  probable  that 
Kutscher  met  with  this  body  although  he  gave  it  the  formula 
CgHgN^O^  in  the  auto-digestion  of  yeast.  Uracil  has  been  synthesized 
by  Fischer  and  Roeder.* 

Uracil  crystallizes  in  needles,  arranged  in  rosettes.  It  does  not 
sublime  as  easily  as  does  thymin.  The  synthetic  product  on  rapid 
heating  becomes  brown  at  280°  and  melts  at  about  335°  with  evo- 
lution of  gas.  It  is  easily  soluble  in  hot,  rather  difficultly  in  cold 
water  and  is  almost  insoluble  in  alcohol  and  in  ether.  It  is  easily 
soluble  in  ammonia  but  it  does  not  unite  with  hydrochloric  or  with 
nitric  acids.  When  treated  in  the  same  way  as  thymin  it  yields 
Weidel's  reaction.  It  therefore  in  all  probability  possesses  the  struc- 
ture, as  given  above,  in  which  case  it  is  2-6  dioxypyrimidin. 

A  Body,  CgHgN^O^,  was  isolated  by  Kutscher  ^  from  yeast  under- 
going sterile  auto-digestion.  It  was  precipitated  from  the  ammoni- 
acal  silver  filtrate,  after  removal  of  purin  bases,  on  neutralization 
with  nitric  acid.  When  purified,  however,  the  substance  is  not  pre- 
cipitable  by  silver  nitrate  in  acid  solution  and  is  thrown  down  by  an 
ammoniacal  silver  solution  but  the  precipitate  is  soluble  in  excess  of 
ammonia  and  in  nitric  acid.  After  treatment  with  boiling,  dilute 
nitric  acid  it  is  precipitated  by  silver  nitrate.  The  amorphous  pre- 
cipitate thus  obtained  is  soluble  in  boiling  nitric  acid  and  separates 
out  on  cooling  as  fine  needles. 

The  substance  is  neutral  in  reaction  and  is  precipitated  by  phos- 

1  Berkhte,  34,  1246. 
'Baichte,  34,4178. 
'Berichte.,  34,  2812,  3362,  3956. 
*  Berichte,  34:,  3751,  4129. 
5  Zeits.  physiol.  Chem. ,  32,  67. 


PYRIMIDIN  GROUP.  419 

photungstic  acid.  Inasmuch  as  Levene  obtained  uracil,  C^H^N^Oj, 
in  a  similar  auto-digestion  of  pancreas  it  may  be  that  Kutscher's 
body  has  a  simpler  formula  than  that  given  above  and  moreover  it 
may  be  related  to,  if  not  identical  with,  uracil.  Its  behavior  to 
silver  is  very  much  like  that  of  thymin. 

The  pyrimidin  bodies  may  be  obtained  by  the  hydrolysis  of  pure 
nucleinic  acid  but  the  better  procedure  is  to  separate  them  from  the 
hydrolytic  products  of  the  original  tissue  or  material.  The  method 
perfected  by  Jones  is  especially  useful  for  this  purpose.  He  em- 
ployed the  testicles  of  the  herring  from  which  the  protamin  had 
been  pre\nously  removed  with  an  acid.  This  material  was  mixed 
with  20  per  cent,  sulphuric  acid  and  the  mixture  was  autoclaved  for 
2  hours  at  150°.  This  heating  completely  destroys  the  adenin  but 
is  without  effect  on  the  thymin.  After  removal  of  the  humus  sub- 
stances by  filtration,  powdered  barium  hydrate  is  added  till  a  slight 
but  distinct  alkaline  reaction  is  obtained.  The  precipitate  of  barium 
sulphate  is  filtered  off  and  thoroughly  washed  with  boiling  water. 
The  combined  filtrates  are  then  concentrated  so  that  for  each  100  g. 
of  dry  testicles  there  is  500  c.c.  of  liquid. 

A  preliminary  test  is  now  made  to  ascertain  the  exact  amount  of 
silver  nitrate  necessary.  For  this  purpose,  a  small  but  known 
amount  of  the  liquid  is  taken  and  after  slightly  acidulating  a  2  per 
cent,  solution  of  silver  nitrate  is  added  from  a  burette  till  a  drop  of 
the  liquid  added  to  an  excess  of  baryta  water  yields  a  yellow  and  not 
a  white  precipitate.  The  amount  of  silver  solution  necessary  for 
portions  of  500  c.c.  of  the  liquid  can  then  be  calculated. 

The  original  liquid  is  then  slightly  acidulated  with  nitric  acid 
and  divided  into  portions  of  500  c.c.  each.  To  each  of  these  the 
requisite  amount  of  silver  nitrate  solution  is  then  added.  After 
removal  of  the  precipitate,  the  filtrate  is  rendered  slightly  alkaline 
with  baryta.  This  precipitate  is  washed  by  decantation  and  finally 
drained  over  a  pump.  It  is  then  decomposed  with  hydrogen  sul- 
phid,  under  pressure.  The  united  filtrates  on  evaporation  yield 
crystals  of  thymin.  These  are  decolored  with  charcoal  and  recrys- 
tallized  from  hot  water.  In  the  case  of  herring  testicles  the  yield 
of  thymin  is  about  2  per  cent. 

It  has  already  been  pointed  out  that  the  pyrimidin  bodies  may  be 
considered  as  antecedents  of  the  purin  bodies  formed  in  the  animal 
organism  or  more  correctly  as  cleavage  products  of  the  latter.  The 
relation  of  the  pyrimidin  and  purin  bodies  can  best  be  shown  by 
equations  representing  the  synthesis  of  uric  acid.  Behrend  and 
Roosen  prepared  uric  acid  by  combining  urea  with  a  pyrimidin 
body  —  iso-dialuric  acid. 


420  CHEMISTRY  OF  THE  LEUCOMAINS. 

HN— CO  NH,  HN— CO 

OC     COH     +     CO       =     OC     C— NH    +     2H,0. 

II  I  111?° 

HN— COH  NH,  HN— C— NH 

Iso-DiALUKic  Acid.  Uric  Acid. 

Fischer  and  Ach  prepared  uric  acid  from  the  pyrimidin  body 
pseudo-uric  acid.  This  on  fusion  with  oxalic  acid  or  better  on  boil- 
ing with  20  per  cent,  hydrochloric  acid  yields  uric  acid. 

HN— CO  HN— CO 

OC     CH.NH.CO.NH5    =     OC    C— NH    -f     H,0. 

HN— CO  HN— C— NH 

Pseudo-uric  Acid. 

The  interesting  syntheses  of  uric  acid  and  of  the  purin  bases  by 
Traube  (1900)  are  also  affected  through  the  intermediate  formation 
of  pyrimidin  bodies. 

Episarkin,  C^HgNgO. — This  base  was  isolated  by  Balke  in  1893 
from  urine.  From  about  1,600  liters  of  urine  only  about  0.4 
g.  was  obtained.  It  accompanied  the  hypoxanthin  silver  nitrate. 
The  mixture  on  digestion  with  dilute  ammonia  on  a  water-bath 
gave  hypoxanthin-silver,  whereas  episarkin,  in  part,  passed  into 
solution.  The  solution  was  filtered,  and  the  slightly  ammoniacal 
filtrate  on  standing  for  twelve  hours  gave  a  crop  of  small  needles 
of  episarkin.  The  hypoxanthin-silver  still  contained  an  admixture 
of  the  new  base,  as  was  found  by  decomposition  with  hydrogen 
sulphid  and  concentration. 

The  crystals  are  more  difficultly  soluble  in  water  and  their 
crystalline  form  is  distinctly  different  from  that  of  hypoxanthin.  A 
separation  can  best  be  accomplished  by  dissolving  the  mixture  of  the 
two  bases  in  as  little  dilute  ammonia  as  possible,  then  on  saturating 
this  solution  with  carbonic  acid  small  whetstone-shaped  needles 
separate.  In  this  respect  it  resembles  epiguanin.  When  recrystal- 
lized  from  hot  water  it  yields  small  prisms  or  needles  which  may 
attain  a  length  of  1  cm.  and  may  be  grouped  in  bunches.  They  are 
white  and  glassy,  and  when  dry  form  a  felt-like  mass.  The  crystals 
are  permanent  and  do  not  effloresce. 

On  evaporation  with  concentrated  nitric  acid,  it  gives  a  yellow 
residue  which  with  sodium  hydrate  becomes  paler.  On  evaporation 
with  nitric  acid  and  chlorin  water  (Weidel's  reaction),  it  gives  a 
white  residue  unaltered  by  ammonia.  When  evaporated  on  a  water- 
bath  with  hydrochloric  acid  and  potassium  chlorate  it  leaves  a  white 
residue,  which  becomes  colored  an  intense  violet  by  ammonia.  This 
reaction  certainly  shows  that  this  base  is  related  either  to  the 
purin  or  to  the  pyrimidin  bodies. 


EPISABKIN.  421 

It  is  difficultly  soluble  in  hot  water ;  almost  wholly  insoluble  in 
cold  water  (1  :  13,000).  It  is  readily  soluble  in  dilute  hydrochloric 
acid,  from  which,  on  evaporation,  the  hydrochlorid  crystallizes  in 
easily  soluble,  pretty  needles.  The  base  does  not  yield  an  insoluble 
compound  with  sodium  hydrate,  though  it  is  possible  that  the  whet- 
stone-shaped crystals  mentioned  above  represent  an  ammonium  salt. 
With  silver  nitrate  it  gives  a  precipitate  which  is  insoluble  in  nitric 
acid,  easily  soluble  in  ammonia.  In  other  respects  the  salt  resembles 
that  of  hypoxanthin.  Thus,  by  boiling  with  nitric  acid  (1.1  sp.  g.) 
a  crystalline  silver  nitrate  compound  forms  which  probably  contains 
one  atom  of  silver,  and  on  digestion  with  dilute  ammonia  yields  a 
silver  compound  with  two  atoms  of  silver — since  a  part  of  the  epi- 
sarkin  passes  into  the  filtrate  and  crystallizes  out  on  cooling.  The 
base  gives  white  precipitates  with  phosphotungstic  acid,  mercuric 
chlorid,  and  ammoniacal  lead  acetate. 

It  is  distinguished  from  adenin  and  hypoxanthin  by  its  almost 
complete  insolubility  in  cold  water ;  from  the  latter  by  not  being 
precipitated  by  picric  acid,  and  by  not  giving  the  characteristic  ruby- 
red  coloration  in  the  alkalinized  filtrate,  after  reduction  with  zinc  and 
hydrochloric  acid  (page  370).  From  xanthin  it  is  distinguished 
by  the  absence  of  the  xanthin  reaction ;  from  hetero-  and  paraxan- 
thin  by  the  absence  of  the  insoluble  sodium  compound  ;  from  guanin 
and  adenin  by  not  clouding  at  53°,  and  by  the  picric  acid  reaction. 
The  fact  that  it  is  not  precipitated  by  picric  acid  and  is  precipitated 
by  ammoniacal  lead  acetate  distinguishes  it  from  epiguanin. 

Kriiger,  in  1895,  from  tea  extract  isolated  a  base  resembling 
somewhat  episarkin.  It  differed,  however,  from  the  latter  in  giving 
with  picric  acid  a  very  fine  crystalline  compound.  It  was  more  soluble 
in  water,  and  gave  different  color  reactions. 

Nothing  definite  can  be  stated  regarding  the  constitution  of  epi- 
sarkin. The  formula  as  given  by  Balke  is  open  to  the  objection  that 
the  sum  of  the  hydrogen  and  nitrogen  atoms  is  an  odd  number. 
Changing  for  this  reason  the  number  of  hydrogen  atoms  to  five,  we 
would  then  have  a  compound,  C^H^NgO.  Such  a  body  may  be 
looked  upon  as  an  amino  derivative  of  uracil  to  which  it  would 
bear  the  same  relation  that  guanin  bears  to  xanthin.  This  relation- 
ship to  the  pyrimidin  group  would  be  expressed  by  the  structural 
formula  : 

HN— CO 

NH,.C 


According  to  this  structure  episarkin  with  nitrous  acid  should 
yield  uracil  and  on  cleavage  should  give  guanidin.  Furthermore, 
it  would  appear  to  be  derived  from  guanin.  Thymin  itself  may  be 
looked  upon  as  a  cleavage  and  oxidation  product  of  epiguanin. 


422  CHEMISTRY  OF  THE  LEUCO MAINS. 

A  Base,  C^H^NgO,  was  obtained  by  Gautier  from  fresh  muscle 
tissue  of  beef,  according  to  the  method  given  on  page  455,  and  on 
account  of  a  resemblance  in  some  of  its  properties  with  xanthin  he 
named  it  pseudoxanthin.  This  name  is  very  inappropriate,  not 
only  because  it  differs  so  much  in  its  empirical  formula  from  that  of 
xanthin,  CgH^N^Oj,  but  also  because  the  term  pseudoxanthin  has 
already  been  applied  by  Schultzen  and  Filehne  to  a  body  isomeric 
with  xanthin,  which  was  obtained  by  the  action  of  sulphuric  acid 
on  uric  acid. 

The  free  base  forms  a  light  yellow  powder,  slightly  soluble  in  cold 
water,  soluble  in  weak  alkali  and  in  hydrochloric  acid.  The  hydro- 
chlorid  is  very  soluble,  and  it  forms  stellate  prisms  with  curved 
faces,  which  resemble  the  corresponding  salt  of  hypoxanthin,  and  to 
some  extent,  also,  the  whetstone-shaped  crystals  of  uric  acid. 

Like  xanthin,  its  aqueous  solution  is  precipitated  in  the  cold  by 
mercuric  chlorid,  silver  nitrate,  and  by  ammoniacal  lead  acetate,  but 
not  by  normal  lead  acetate.  On  evaporation  with  nitric  acid,  the 
residue  gives,  on  contact  with  potassium  hydrate,  as  in  the  case  of 
xanthin,  a  beautiful  orange-red  coloration  (xanthin  reaction).  It 
differs  from  xanthin,  not  only  in  its  empirical  composition,  but  also 
in  its  greater  solubility  and  in  its  crystalline  form.  It  is  possible 
that  this  base,  on  account  of  its  great  resemblance  to  xanthin,  may 
have  been  mistaken,  at  different  times,  for  that  compound.  It  is  to 
be  noted,  however,  that  this  base  has  not  been  met  since  Gautier'e 
work.  If  the  formula  given  is  correct,  the  base  may  be  considered 
as  a  pyrimidin  body,  such  as  a  di-imido  derivative  of  the  preceding. 

HEXON  BASES. 

The  purin  bases  through  the  studies  of  Kossel  have  been  shown 
to  be  derivatives  of  the  nuclein  which  exist  within  the  nuclei  of  cells. 
They  are  therefore  to  be  looked  upon  as  essentially  products  of  nuclear 
metabolism  and  more  especially  of  the  nucleinic  acids.  If  the  results 
obtained  by  Bang  with  guanylic  acid,  a  nucleinic  acid  from  the  pancreas, 
be  correct  and  hold  true  for  the  other  nucleinic  acids  then  all  the  nitro- 
gen contained  in  these  acids  is  in  the  purin  form  and  hence  would  leave 
the  body  to  some  extent  as  purin  bases,  but  chiefly  in  the  oxidized 
form  as  uric  acid.  A  portion  of  the  purin  bodies,  however,  may 
undergo  hydrolytic  change  and  appear  in  the  urine  as  pyrimidin 
derivatives  or  even  in  the  more  simple  form  of  allantoin,  creatinin  and 
urea.  The  presence  of  allantoin  and  oxalic  acid  in  urine  and  even 
in  sprouts  is  thus  accounted  for. 

On  the  other  hand  extensive  investigations  carried  on  during  the 
past  ten  years  have  shown  the  existence  of  a  group  of  basic  sub- 
stances characteristic  of  proteid  bodies  and  in  no  wise  related  to  the 
nucleinic  acids  and  their  products.     These  basic  substances  have 


HEXON  BASES.  423 

been  designated  by  Kossel  as  the  hexon  bases  inasmuch  as  they  con- 
tain six  carbon  atoms. 

Arginin  the  first  known,  and  perhaps  the  most  important  member 
of  this  group,  was  discovered  by  Schulze  and  Steiger^  in  1886  in 
lupine  sprouts  wherein  it  is  formed  as  a  result  of  the  energetic  pro- 
teid  disintegration  which  takes  place  in  the  developing  plant.  The 
base  exists  preformed  in  the  cotyledons  inasmuch  as  it  can  be  ex- 
tracted from  these  by  water  alone  without  the  aid  of  hydrolytic 
agents.  In  this  way  the  dry  sprouts  yielded  as  much  as  3.5  per 
cent,  of  arginin.  Nine  years  later  Hedin^  demonstrated  that 
arginin  could  be  obtained  by  the  hydrolytic  cleavage  of  a  number 
of  proteid  substances.  On  decomposing  these  by  boiling  with  hy- 
drochloric acid  and  tin,  he  obtained  variable  amounts  of  the  base  as 
shown  below  and  at  no  time  was  the  quantity  as  large  as  that  ob- 
tained by  Schulze  from  sprouts. 

The  yield  was  as  follows  : 

Conglutin,        2.75  per  cent.  Egg  albumin,  0.8  per  cent. 

Gelatin,  2.6       "      "  Blood  serum,  dried,  0.7    "      " 

Horn,  2.5       "      "  Casein,  0.25"      " 

Yolk  albumin,  2.3      "      " 

As  yet  no  proteid  is  known  which  does  not  yield  arginin  on 
hydrolysis.  Elastin  which  was  studied  by  Berg  and  by  Hedin  was 
supposed  to  be  free  from  arginin  but  Kossel  and  Kutscher  ^  showed 
by  means  of  improved  methods,  that  it  did  contain  about  0.3  per 
cent,  of  the  base.  Subsequently  lysin  was  also  shown  to  be  present. 
Of  the  total  nitrogen  contained  in  the  young  sprouts  of  pine  and  fir 
21  to  29  per  cent.,  according  to  Schulze,^  is  represented  by  arginin 
while  the  proteid  from  the  seeds  of  the  same  plants  on  cleavage  with 
hydrochloric  acid  and  tin  yield  as  much  as  10  per  cent,  of  the  base. 
On  a  subsequent  occasion  a  yield  of  only  6  per  cent,  was  obtained 
from  fir  seeds.  The  proteids  contained  in  the  seeds  of  plants  are 
apparently  extremely  rich  in  arginin  which  is  set  free  during  the 
active  proteid  destruction  which  ensues  during  germination.  This 
becomes  readily  comprehensible  when  it  is  remembered  that  tryptic 
ferments  are  brought  into  being  in  the  process  of  germination 
(Butkewitch)  and  that  the  hydrolytic  cleavage  which  these  ferments 
induce  is  analogous  to  that  produced  by  acids,  and  corresponds  to 
the  pancreatic  digestion  of  proteids.  This  fact  will  be  rendered  the 
more  apparent  on  comparison  of  the  digestion  products  formed  by 
trypsin  and  the  following  bodies  which  Schulze  ^  has  isolated  and 

^Zeits.  physiol.  Chem.,  11,  44  (1886). 

^Zeits.  physiol.  Chem.,  21,  155  (1895);  20,  186  (1894). 

^Zeits.  physiol.  Chem.,  25,  551  (1898)  ;  31,  205. 

*Zeits.  physiol.  Chem.,  22,  435  ;  25,  360. 

*  Zeits.  physiol.  Chem. ,  28,  470. 


424  CHEMISTRY  OF  THE  LEUCOMAINS. 

shown  to  exist  preformed  in  germinating  plants  :  Leucin,  tyrosin, 
phenyl  alanin,  amido  valerianic  acid,  asparagin,  glutamin,  arginin, 
histidin  and  lysin. 

The  three  last  mentioned  substances,  the  hexon  bases,  have  been 
only  recently  shown  to  be  products  of  tryptic  digestion.  Indeed, 
the  studies  of  Kutscher  ^  have  shown  that  the  so-called  antipepton  is 
not  a  chemical  substance  possessing  a  definite  formula,  Cj^H^gNgOj, 
as  Siegfried  believes,  but  is  rather  a  heterogeneous  mixture  from 
which  he  was  able  to  isolate  leucin,  tyrosin,  aspartic  and  glutamic 
acids,  arginin,  histidin  and  lysin.  In  other  words  trypsin,  like  sul- 
phuric acid,  if  given  sufficient  time  will  completely  split  up  the  pro- 
teid  molecule  and  will  not  stop  short,  as  has  been  heretofore  held 
with  a  resistant  antipepton.  An  exactly  similar  condition  is  met 
with  in  the  auto-digestion  of  yeast  where  not  only  xanthin  bases  and 
amido  acids,  but  also  hexon  bases  form  (Kutscher^).  It  is  of  interest 
to  note  that  according  to  Levene  ^  arginin,  histidin  and  uracil,  and 
possibly  thymin,  are  formed  during  the  auto-digestion  of  the  pancreas. 
From  the  liver  of  a  case  of  acute  yellow  atrophy  Taylor  obtained 
leucin  and  asparaginic  acid  but  no  hexon  or  purin  bases.  Hedin  * 
was  the  first  to  point  out  the  presence  of  lysin  in  tryptic  digestion  of 
fibrin  and  Kossel  and  Mathews  found  the  three  hexon  bases  in  the 
similar  digestion  of  sturin. 

The  investigations  of  Lawrow  ^  show  that  the  proteid  molecule  is 
also  destroyed  in  peptic  digestion.  Thus  in  the  sterile  auto-digestion 
of  pigs'  stomachs  he  obtained  leucin,  asparaginic  and  amido  valerianic 
acids  but  no  tyrosin.  Hexon  bases  were  not  present  but  instead 
large  amounts  of  cadaverin  and  putrescin.  The  latter  two  bases 
undoubtedly  resulting  by  the  pepsin  hydrolysis  of  the  proteid.  A 
somewhat  similar  result  was  obtained  by  Kutscher  ^  in  the  auto-diges- 
tion of  the  thymus  gland.  Of  still  greater  significance  is  the  observa- 
tion of  Kutscher  and  Seemann''  that  in  the  small  intestines  albu- 
moses  and  peptons  are  nearly  absent  and  that  leucin,  tyrosin,  lysin 
and  arginin  are  present  as  a  result  of  energetic  tryptic  action. 

Haslam  ^  has  shown  that  the  three  bases  exist  in  different  amounts 
in  deutero-  and  hetero-albumose.  Hart^  found  that  the  yield  of 
hexon  bases  was  about  the  same  in  syntonin  and  protalbumose  whereas 
in  heteroalbumose  the  relative  amounts  were  different  and  to  a  cer- 
tain extent  depended  upon  the  quantity  of  salt  present. 

According  to  Emmerling  ^°  the  enzyme  papayotin  acting  on  blood 

^Zeils.  physid.  Chem.,  25,  195  ;  26,  110  ;  28,  88  (1899). 

^  Zeits.  physiol.  Chem.,  32,  59. 

^Zeits.  physiol.  Chem.,  32,  540  (1901). 

*Zeiis.  phydol.  Chem.,  21,  298. 

s^eite.  phydol.  Chem.,  26,  512  ;  33,  312. 

^ Zeits.  physiol.  Chem.,  34,  117. 

''  Zeits.  physiol.  Chem.,  34,  543. 

^ Zeits.  physiol.  Chem.,  32,  54. 

^ Zeits.  physiol.  Chem.,  33,  347. 

^°Berichte,  35,  695,  700  (1902). 


HEXON  BASES.  425 

fibrin  in  the  presence  of  toluol  yields  not  only  albumose  and  pepton 
but  also  simpler  cleavage  products  such  as  arginin,  leucin,  tyrosin, 
glycocoll,  alanin,  phenyl  alanin,  asparaginic  and  glutamic  acids.  The 
enzyme  of  B.  fluorescens  liquefaciens  under  like  conditions  gave 
arginin,  leucin  and  asparaginic  acids. 

It  is  evident  from  what  has  been  said  that  arginin  and  the  other 
hexon  bases  exist  preformed  in  the  proteid  molecule  and  that  they 
are  liberated  or  split  oif  by  the  action  of  acids  and  of  tryptic  enzymes 
and  by  oxidation  with  potassium  permanganate  (Jolles).  A  most 
interesting  question  arises  as  to  whether  these  hexon  bases  are  also 
liberated  in  the  tissue  metabolism  of  the  body.  Already  Gule- 
witsch  ^  has  shown  that  the  spleen,  on  standing  with  thymol  for 
eighteen  hours,  contains  arginin.  This  may  be  due  to  initial  hy- 
drolysis, especially  since  it  is  known  that  the  spleen  and  other  organs 
of  the  body  contains  an  active  proteolytic  enzyme  (Hedin  and  Row- 
land).^ As  yet  the  hexon  bases  have  not  been  detected  in  the  urine, 
but  it  is  not  unreasonable  to  suppose,  inasmuch  as  they  are  unques- 
tionably antecedents  of  urea,  that  they  will  appear  in  the  urine  when 
normal  urea  formation  is  interfered  with.  The  marked  resemblance 
which  arginin  bears  to  creatinin  and  to  the  leucoraains  of  that  group 
was  pointed  out  from  the  first  by  Schulze. 

The  second  member  of  the  hexon  group,  lysin,  we  owe  to  the 
brilliant  investigation  of  DrechseP  (1890)  on  the  cleavage  products 
of  casein.  On  boiling  casein  with  hydrochloric  acid  and  tin  he  ob- 
tained ammonia,  amido  acids,  and  two  bases  lysin  and  lysatinin. 
Fischer,  a  pupil  of  Drechsel,  obtained  the  same  products  by  hydro- 
lyzing  gelatin  in  like  manner  (Inaug.  Diss.  1890).  Subsequently, 
Siegfried*  showed  that  a  number  of  proteids,  conglutin,  gluten- 
fibrin,  hemiprotein,  oxyprotosulfonic  acid  and  egg  albumin  also 
yielded  the  two  bases  found  by  Drechsel.  Hedin  isolated  lysin 
from  horn,  casein,  conglutin,  egg  albumin,  yolk  albumin,  and  blood 
serum.  By  employing  the  same  method  Schwarz*  obtained  from 
the  elastin  of  the  aorta  some  lysatinin.  In  1895,  however,  Hedin* 
showed  that  lysatinin  was  really  a  mixture  of  equal  molecules  of 
lysin  and  arginin.  Since  then  lysin  has  been  found  associated  with 
arginin  and  histidin  in  various  proteids  of  animal  origin  with  the 
exception  of  the  several  protamins  indicated  in  the  subjoined  table. 
Its  presence  in  "  antipepton  "  and  in  tryptic  digestion  of  fibrin  and 
sturin  has  already  been  referred  to.  Schulze'^  rounded  out  his 
studies  on  arginin  in  plants  by  showing  that  both  lysin  and  histidin 
were  also  present  in  lupine  sprouts  and  in  the  seeds  of  the  fir.     The 

>  Zeiis.  physiol.  Chem.,  30,  533  (1900). 

^Zeits.  physiol.  Chem.,  32,  341,  531,  1901. 

^Berkhte,  23,  3096. 

*Berichte,  24,418,  1891. 

^Zdts.  physiol.  Chem.,  18,  487. 

^Zeits.  physiol.  Chem.,  21,  297,  1895  ;  20,  186, 

''  Zeits.  physiol.  Chem.,  28,  459,  465. 


426 


CHEMISTRY  OF  THE  LEUCOMAINS. 


three  bases  therefore  result  as  a  rule  simultaneously  in  the  hydro- 
lytic  cleavage  of  proteids  irrespective  as  to  whether  this  is  induced  by 
acids  or  by  enzymes.  From  the  table  given  below  it  will  be  seen 
that  lysin  is  not  only  absent  from  several  protamins  but  also  from 
the  alcohol  soluble  proteids  of  wheat  and  maize.  In  the  latter  case 
it  is  noteworthy  that  the  absence  of  lysin  is  associated  with  an  un- 
usually large  amount  of  ammonia.  Levene  and  Mendel/  it  should 
be  noted,  obtained  by  hydrolysis  of  edestin,  the  crystalline  proteid 
from  hemp-seed,  arginin,  histidin  and  undoubtedly  lysin.  Accord- 
ing to  Schulze  and  Winterstein  ^  edestin  and  the  proteid  from  seeds 
of  conifers  yields  more  arginin  than  do  other  vegetable  proteids. 
Edestin  gave  11  per  cent,  arginin;  1.17  per  cent,  histidin  and  1.3 
per  cent,  lysin. 

The  splendid  researches  of  Kossel  ^  on  the  protamins  and  their 
cleavage  products  led  to  the  discovery  of  histidin,  the  third  hexon 
base.  Undoubtedly,  histidin  was  obtained  by  Siegfried  in  his  studies 
on  the  cleavage  products  of  proteids  (1891),  but  it  remained  for 
Hedin  *  to  clearly  demonstrate  its  presence  among  the  hydrolytic 
products  of  casein,  egg  albumin,  blood  serum  and  horn.  Since  then 
it  has  been  found  as  a  constant  cleavage  product  of  the  more  com- 
plex animal  and  plant  proteids. 

Recently  Kossel  and  Kutscher  ^  have  carried  out  a  most  careful 
study  upon  the  quantitative  distribution  of  the  hexon  bases  in  the 
different  proteids.  Their  results  in  per  cent,  are  given  in  the  fol- 
lowing table  : 


'53 

1 

if 

o  g 

.2 

d 

•s 

1 

.2 

d" 

a' 

"3 

CO 

s 

3q 

H 

Wo 

"3 

O 

5 

.2 

5 

3 

tsi 

Arginin. 

84.3 

82.2 

62.5 

58.2 

14.36 

15.52 

9.3 

4.4 

3.05 

3.13 

2.75 

1.82 

Lysin. 

0 

0 

0 

12.0 

7.7 

8.30 

5-6 

2.15 

0 

0 

0 

0 

Histidin. 

0 

0 

0 

12.9 

1.21 

2.34 

small 

1.16 

1.53 

0.43 

1.20 

0.81 

Ammonia. 

0 

0 

— 

0 

1.66 

0.74 

0.3 

2.45 

3.89 

4.23 

4.1 

2.56 

Arginin,  it  will  be  seen,  is  a  constant  constituent,  and  is  by  far  the 
most  abundant  of  the  hexon  bases.  The  amounts  found  in  several 
instances  are  higher  than  those  which  Hedin  obtained  (p.  423). 
Hedin's  results  represent  the  minimum  values  obtained  by  a  some- 
what imperfect  method.  For  the  amounts  of  arginin  in  seeds,  sprouts 
and  in  elastin  see  p.  423. 

^  Am.  Journ.  Physiol.,  6,  48,  1901. 
^  ZeiUi.  physiol.  Chem.,  33,  557. 
'Zette.  'phydol.  Chem.,  22,  178,  1896. 
*  Zeits.  physiol.  Chem.,  22,  191. 
5  Zeit8.  physiol.  Chem.,  31,  207,  1900. 


HEXON  BASES.  427 

The  method  which  Kossel  and  Kutscher  >  have  employed  for  the  isola- 
tion and  separation  of  the  hexon  bases  in  a  very  condensed  form  is  as  fol- 
lows :  For  every  gram  of  proteid  3  c.c.  of  concentrated  H,S04  and  6  c.c. 
of  water  are  taken  and  the  mixture  is  boiled  for  14  hours  under  an  inverted 
condenser.  The  liquid  is  then  made  up  to  a  definite  volume  and  the  total 
nitrogen  in  a  definite  portion  can  be  determined.  Hot,  saturated  baryta 
is  then  added  till  only  a  slight  acidity  remains,  after  which  the  BaSO^  is 
drained  and  thoroughly  washed.  The  filtrate  and  wash-water  are  com- 
bined and  the  total  nitrogen  in  a  portion,  is  again  determined.  The 
difference  represents  the  nitrogen  in  the  humin  substances,  which  are 
dragged  down  with  the  BaSOi. 

In  another  portion  the  ammonia  is  determined  by  distillation  with  mag- 
nesia. The  ammonia  is  expelled  from  the  remaining  total  fluid  by  evap- 
oration with  magnesia.  The  combined  ammonia-free  fluids  are  then  ren- 
dered strongly  alkaline  with  baryta  and  the  resultant  precipitate  is 
thoroughly  washed.  The  filtrate  and  wash-water  are  combined  and  acid- 
ulated with  HjSO^.  The  precipitate  which  forms  is  likewise  thoroughly 
washed  and  the  liquids  are  united,  made  up  to  a  definite  volume  and  the 
total  nitrogen  in  a  portion  again  determined  =  Humin  nitrogen  II. 

To  separate  the  bases  present  arginin  and  histidin  are  precipitated  to- 
gether, while  the  lysin  remains  in  solution.  For  this  purpose  the  acid 
liquid  is  diluted,  heated  on  the  water-bath  and  silver  sulphate  is  added 
till  a  drop  of  the  liquid  added  to  an  excess  of  baryta  yield  a  yellow  pre- 
cipitate. When  this  point  is  reached  the  liquid  is  cooled  to  40°  and  sat- 
urated with  baryta.  The  precipitate  of  arginin-  and  histidin-silver  is 
removed  at  once  and  washed  thoroughly  with  baryta. 

To  separate  the  arginin  and  histidin,  the  precipitate  is  suspended  in 
dilute  sulphuric  acid  and  decomposed  with  hydrogen  sulphid.  The  sul- 
phid  precipitate  is  removed  and  thoroughly  washed.  The  combined  fil- 
trates are  evaporated  to  expel  the  gas,  then  made  up  to  a  definite  volume 
and  the  total  nitrogen  in  a  portion  is  determined.  The  solution  is  then 
neutralized  with  baryta,  after  which  barium  nitrate  is  added  till  all  the 
sulphuric  acid  is  precipitated.  The  precipitate  is  removed  and  well  washed. 
The  filtrates  are  united,  concentrated  to  about  300  c.c,  after  which  silver 
nitrate  is  added  until  a  drop  of  the  liquid  when  added  to  an  excess  of 
baryta  gives  a  yellow  precipitate.  The  solution  is  carefully  neutralized 
with  baryta,  after  which  the  reagent  is  added  from  a  burette  in  small  por- 
tions at  a  time  till  all  the  histidin  has  been  precipitated.  This  point  is 
reached  when  a  portion  of  the  liquid  slowly  added  to  weak  ammoniacal 
silver  nitrate  yields  no  precipitate.  If  a  precipitate  forms  and  is  soluble 
in  excess  of  ammonia,  histidin  is  present. 

The  histidin-silver  precipitate  is  filtered  off  and  washed  well.  The  fil- 
trates contain  arginin.  To  separate  the  histidin  the  precipitate  is  suspended 
in  water,  acidulated  with  HjS04,  and  decomposed  with  hydrogen  sulphid. 
The  combined  filtrate  and  wash-water  are  then  freed  from  sulphuric  acid 
by  means  of  baryta.  The  latter  is  removed  and  the  final  filtrate  is  evap- 
orated to  dryness.  The  residue  is  taken  up  with  silver  nitrate  solution  to 
which  a  drop  of  nitric  acid  was  added  and  the  solution  is  filtered.  The 
filtrate  on  evaporation  yields  the  crystalline  histidin  dichlorid,  which  is  dried 
in  a  vacuum  at  40°  and  weighed. 

The  above  arginin  filtrate  is  saturated  with  powdered  baryta  and  the 
resultant  precipitate  is  well  washed,  after  which  it  is  suspended  in  acidu- 
lated water  and  decomposed  with  hydrogen  sulphid.  The  combined  fil- 
trates are  evaporated  and  then  made  up  to  a  definite  volume.  The  nitro- 
gen in  a  portion  is  determined  and  the  total  amount  of  arginin  is  calcu- 

^Zeits.  physiol.  Chem.,  31, 166-178  ;  25,  178. 


428  CHEMISTRY  OF  THE  LEUCOMAINS. 

lated.  The  liquid  is  then  freed  from  acid  by  baryta  and  from  the  latter  by 
carbonic  acid,  after  which  it  is  neutralized  with  nitric  acid  and  evaporated. 
When  dried  in  a  vacuum  it  can  be  weighed  as  the  neutral  nitrate  CgHj^ 
N4O3.HNO3  +  JH2O.  The  arginin  may  also  be  estimated  polarimetrically, 
or  as  the  acid  di-nitrate. 

The  lysin  is  contained  in  the  original  filtrate  from  the  histidin  and 
arginin  silver  precipitate.  To  isolate  it,  the  solution  is  acidulated  and  the 
silver  removed  with  hydrogen  sulphid.  The  combined  filtrate  and  wash- 
water  is  concentrated  to  about  500  c.c,  and  after  sulphuric  acid  is  added 
to  make  a  5  per  cent,  solution  it  is  precipitated  with  phosphotungstic 
acid.  The  precipitate  is  thoroughly  washed.  It  contains  most  of  the 
lysin  and  a  small  amount  of  mono-amido  acids.  The  bulk  of  the  latter  is 
contained  in  the  filtrate.  The  precipitate  is  decomposed  with  baryta  and 
filtered.  The  combined  filtrate  after  removal  of  excess  of  barium  with 
carbonic  acid  is  evaporated  to  dryness.  The  residue  is  taken  up  in  water, 
filtered,  and  the  filtrate  is  again  evaporated  to  dryness.  The  residue  is 
now  taken  up  in  alcohol  and  an  alcoholic  picric  acid  solution  is  added, 
carefully  avoiding  an  excess.  The  precipitate  is  filtered  off",  washed  with 
a  little  absolute  alcohol  and  then  is  dissolved  in  boiling  water.  The  solu- 
tion after  concentration  and  cooling  yields  needle-shaped  crystals  which 
are  filtered  ofif,  washed  and  weighed  as  lysin  picrate.  Some  lysin  remains 
in  the  mother  liquors. 

Instead  of  decomposing  the  phosphotungstic  acid  precipitates  witb  baryta 
Winterstein  advises  extraction  with  ether  and  hydrochloric  acid  {Z.  P.  C. , 
34,  154). 

It  should  be  noted  in  this  connection  that  the  amount  of  lysin  and  or 
ammonia  in  proteid  cleavage  depends  upon  the  amount  of  salt  present. 
When  salt  is  present  the  nitrogenous  humin  substances  are  decreased  and 
the  yield  of  lysin  and  ammonia  is  increased  (Hart,  Z.  P.  C,  33,  347). 

PROTAMINS   AND   HISTONS. 

By  far  the  most  fruitful  result  of  the  study  of  the  hexon  bases  is 
the  light  which  has  been  shed  upon  the  composition  of  the  proteids. 
Moreover  a  better  insight  has  been  gained  into  the  nitrogen  metab- 
olism of  the  body.  The  fact  that  arginin  on  hydration  with  baryta 
yields  urea  indicates  the  existence  of  a  urea  or  rather  guanidin  group 
in  that  base  and  hence  in  every  proteid.  From  the  constitution  of 
arginin  and  lysin  (p.  437,  444)  it  will  be  seen  that  these  represent 
respectively  diamidovalerianic  and  diaraidocaproic  acids.  It  is  by  the 
cleavage  of  these  acids  that  bacteria  (and  enzymes,  Lawrow)  yield 
the  well  known  ptomains  cadaverin  and  putrescin.  The  presence  of 
these  ptomains  in  cystinuria  is  now  more  readily  understood.  The 
monamido  acids  represented  by  leucin,  tyrosin,  asparaginic  acid  and 
phenyl  alanin,  make  up  a  third  type  of  nitrogen  combination  in  the 
proteid  molecule.  The  liberation  of  ammonia  by  hydrolytic  change, 
as  seen  in  the  table  on  p.  426,  indicates  the  existence  of  a  fourth  as 
yet  unknown  nitrogen  group.  The  bulk  of  the  proteid  nitrogen  is 
therefore  found  in  the  four  groups  mentioned.  The  fact  that  am- 
monia and  amido  acids  are  changed  in  the  body  into  urea,  when 
applied  to  the  proteid  molecule  as  thus  understood,  readily  explains 
the  transformation  of  the  greater  part  of  the  nitrogen  of  our  food 
into  the  final  waste  product,  urea. 


PROTAMINS  AND  HISTONS.  429 

The  simplest  group  of  proteids  are  the  protamins.  We  owe  the 
discovery  of  these  bodies  as  well  as  of  nucleins  to  Miescher  ^  who  in 
1874  isolated  the  first  known  representative  of  this  group.  By 
means  of  dilute  hydrochloric  acid  Miescher  extracted  from  the  sper- 
matozoa of  the  salmon  two  new  bodies  protamin,  a  base,  and 
nucleinic  acid.  Piccard  ^  in  the  same  year  extended  somewhat  the 
investigation  of  Miescher  by  ascribing  a  diiferent  formula  and 
demonstrating  the  presence  of  xanthin  bases  in  Miescher's  prep- 
arations. During  the  ensuing  twenty  years  protamin  was  apparently 
wholly  forgotten  and  its  nature  remained  therefore  uncertain.  Since 
1894,  however,  we  know  through  the  labors  of  Kossel  and  his  pupils 
that  Miescher's  substance  is  one  of  a  number  of  closely  allied  bodies. 
For  this  group  KosseP  reserves  the  original  term  protamin  and 
designates  the  individual  compounds  according  to  their  source. 
Thus,  Miescher's  protamin  since  it  is  derived  from  the  salmon  is 
known  as  salmin  ;  that  from  the  sturgeon  becomes  sturin  ;  while  the 
herring  yields  clupein.  To  these  may  be  added  scombrin  *  from  the 
spermatozoa  of  the  mackerel ;  cyclopterin  *  from  the  Cyclopterus 
lumpus  (sea-hare)  ;  aceipenserin  ^  from  Accipenser  stellatus  ;  and 
silurin  from  the  shad  (Silurus  glanis).  From  tubercle  bacilli  Ruppel  "^ 
isolated  a  protamin  to  which  he  has  given  the  name  tuberculosamin. 
A  similar  body  it  may  be  stated  has  been  obtained  from  the  colon 
bacillus  by  Vaughan.  The  closely  related  histon  bodies  are  considered 
on  p.  434. 

From  the  evidence  thus  far  gathered  it  is  clear  that  the  protamins 
are  by  no  means  widely  distributed  in  nature.  As  a  rule  they  are 
present  in  the  mature  spermatozoa  of  fish.  The  immature  sperma- 
tozoa on  the  other  hand  are  believed  to  contain  chiefly  histon.  Ap- 
parently, protamins  are  not  present  in  the  spermatozoa  of  the  higher 
animals.  Miescher  failed  to  find  it  in  those  of  the  steer  and  Math- 
ews^ likewise  failed  to  isolate  it  from  the  steer  and  boar. 

The  one  property  which  is  common  to  all  proteids,  so  far  as  our 
knowledge  at  present  extends,  is  the  formation  of  hexon  bases  on 
cleavage.  From  the  table  on  p.  426  it  will  be  seen  that  arginin  is 
present  in  remarkably  large  quantity  in  the  several  protamins. 
Moreover,  these  bodies  yield  the  biuret  reaction  and  are  converted 
by  trypsin  into  "  protons,"  the  analogues  of  pepton,  and  eventually 
into  the  hexon  bases.  It  is  because  of  these  facts  that  the  protamins 
are  regarded  as  the  simplest  of  proteids.  In  the  spermatozoa  pro- 
tamin undoubtedly  exists  in  a  salt-like  combination  with  nucleinic 

^  Zeits.  physiol.  Chem.,  22,  177. 
■^Berichte,  7,  1714. 

^Zeits.  physiol.  Chem.,  22,  180,  1896. 
^Zeits.  physiol.  Chem.,  26,  524,  1898. 
^Zeits.  physiol.  Chem.,  28,  313,  1899. 
^Zeits.  physiol.  Chem.,  32,  200,  1901. 
T  Zeits.  physiol.  Chem.,  26,  231,  1898. 
^Zeits.  physiol.  Chem.,  23,  399,  1897. 


430  CHEMISTRY  OF   THE  LEUCOMAINS. 

acid.  From  the  investigations  of  Mathews  it  would  seem  that  the 
chromatin  of  these  cells  in  the  case  of  the  herring  is  a  combination 
of  equal    molecules  of  clupein,   CgpHgyNj^Og,   and   nucleinic   acid, 

According  to  Kossel  ^  the  group  or  combination  which  yields  the 
hexon  bases  is  characteristic  of  all  proteids  and  is  therefore  to  be 
considered  as  the  nucleus  of  the  proteid  molecule.  In  the  simplest 
proteids  like  salmin  and  clupein  this  nucleus  yields  but  one  base, 
arginin.  In  others,  as  shown  in  the  table  on  p.  426,  this  nucleus  is 
a  trifle  more  complex  since  it  yields  two  bases,  arginin  and  histidin. 
And  lastly,  as  it  exists  in  the  majority  of  proteids  it  yields  all  three 
hexon  bases. 

The  simplest  protamins  are  rendered  a  trifle  more  complex  by  the 
addition  of  monoamido  acids  to  the  nuclear  group.  Thus,  clupein 
contains  amido  valerianic  acid  but  no  lysin.  Again,  cyclopterin 
unlike  other  protamins  contains  an  aromatic  group  which  is  split  off 
as  tyrosin  (8.3  per  cent.).  The  complexity  of  the  molecule  may 
continue  to  grow  by  the  addition  of  other  monamido  acids  like  leucin, 
glycocoll,  tyrosin,  asparginic  and  glutamic  acid.  Eventually,  the 
molecule  is  increased  by  the  introduction  of  groups  containing  other 
elements,  such  as  sulphur,  iron,  and  even  iodin.  The  growth  of 
more  complex  proteids  is  seen  in  the  fact  observed  by  Kossel  ^  that 
protamins  precipitate  albumin  and  albumoses  and  give  rise  to  histon- 
like  bodies.  Kutscher  has  observed  a  similar  behavior  between 
albumoses  and  other  proteids.  Again,  the  relatively  simple  proteids 
unite  with  entirely  different  groups,  such  as  carbohydrates  yielding 
glycoproteids ;  or  with  nuclein  yielding  nucleoproteids  ;  with  hema- 
tin  giving  rise  to  hemoglobins. 

The  protamins  are  isolated  according  to  the  method  of  KosseP 
For  this  purpose  the  mature  testicles  are  finely  divided,  shaken  up 
with  water  and  strained  through  gauze.  The  milky  liquid  is  pre- 
cipitated by  careful  addition  of  acetic  acid.  The  precipitated  sper- 
matozoa are  boiled  with  alcohol  and  finally  extracted  with  ether. 
500  c.c.  of  a  one  per  cent,  solution  of  sulphuric  acid  are  added  to 
each  100  g.  of  the  dried  spermatic  cells  and  the  mixture  thoroughly 
shaken  for  one  quarter  of  an  hour.  The  liquid  is  filtered  off  and 
the  residue  is  extracted  six  times  in  the  same  way.  The  addition  of 
salt  favors  the  deposition  of  the  precipitate.  The  combined  aqueous 
extracts  are  precipitated  by  the  addition  of  three  volumes  of  alcohol. 
After  decantation  the  precipitate  of  protamin  sulphate  is  drained, 
then  dissolved  in  hot  water  and  re-precipitated  with  alcohol.  To 
remove  traces  of  nucleinic  acid,  the  precipitate  is  dissolved  in  warm 
water    and    reprecipitated  with    sodium    picrate.      The  deposit   is 

^  Zeits.  physwl.  Chem.,  25,  186,  1898. 

*  Zeits.  vhysiol.  Chem.,  22,  178. 

^ZeUs.  physiol.  Chem.,  22, 178  ;  25,  166  ;  32,  198. 


PROTAMINS  AND  HISTONS.  431 

washed  thoroughly,  dissolved  in  sulphuric  acid  and  from  the  solution 
the  picric  acid  is  removed  with  ether.  The  protamin  sulphate  is 
then  thrown  down  with  alcohol  and  the  whole  process  is  repeated  if 
necessary  till  the  substance  forms  a  white  flocculent  precipitate. 

The  protamins  are  strong  bases  and  the  aqueous  solution  possesses 
a  marked  alkaline  reaction.  With  acids  they  form  well  defined 
salts.  The  sulphates  usually  form  a  white  powder  which,  in  the 
case  of  sturin  and  accipenserin,  is  very  soluble  in  water.  The  sul- 
phate of  clupein  is  soluble  in  warm  water  and  separates  out  on  cool- 
ing as  oily  globules.  The  solutions  of  protamins  are  levo-rotatory 
and  give  the  biuret  reaction  in  the  cold.  Cyclopterin  is  the  only 
protamin  which  gives  the  Millon  test  thus  indicating  the  presence  of 
an  aromatic  group.  From  it  Kossel  and  Kutscher  obtained  8.3  per 
cent,  of  tyrosin.  Clupein  gave  amido  valerianic  acid.  In  the  pres- 
ence of  ammonia  they  precipitate  pepton  solutions.  It  is  of  inter- 
est to  note  that  the  sulphates  are  precipitated  by  saturated  salt  or 
ammonium  sulphate  solutions.  The  protamins  are  precipitated  by 
the  usual  alkaloidal  reagents  even  in  neutral  or  slightly  alkaline 
solutions.  In  this  respect  they  differ  from  the  peptons  and  albu- 
moses  which  require  an  acid  reaction.  Sodium  tungstate,  phospho- 
tungstate,  chromate,  ferrocyanid,  picrate  and  solutions  of  iodin  and 
bromin  give  precipitates.  Benzoyl  chlorid,  silver  nitrate  and  mer- 
curic chlorid  likewise  precipitate  the  protamins.  They  are  also 
thrown  down  as  cuprous  compounds  by  copper  sulphate  and  sodium 
hyposulphite. 

On  boiling  with  dilute  sulphuric  acid  for  a  short  time  the  pro- 
tamins hydrolyze  partially,  forming  substances  which  may  be  com- 
pared to  the  peptons.  To  these  primary  cleavage  products  Kossel 
has  applied  the  term  proton.  On  prolonged  boiling  with  acid  these 
are  decomposed,  giving  rise  to  the  hexon  bases.  Kossel  and  Mathews 
have  shown  that  while  pepsin  is  without  effect  on  the  protamins, 
trypsin  decomposes  them,  like  an  acid,  yielding  protons  and  eventu- 
ally the  hexon  bases.  In  this  case  the  biuret  reaction  disappears  as  in 
the  auto-digestion  of  the  pancreas.  In  this  respect  the  digestion  of 
protamins  is  similar  to  that  of  the  more  complex  proteids  (see  page 
424). 

Salmin  and  clupein  according  to  Kossel  ^  are  identical  and  possess 
the  formula  CggHgyNj^Og.  The  composition  of  sturin  is  represented 
by  CggHg^N^gO^  and  that  of  accipenserin  by  Q^^H^^^f)^. 

As  to  the  inner  structure  of  the  protamin  molecule  very  little  that 
is  definite  can  be  said.  The  large  amount  of  arginin  present  in 
these  substances  indicates  that  this  base  forms  an  important  part  of 
the  molecule.  The  hexon  bases  contain  each  6  carbon  atoms  and 
the  comparison  of  these  by  Kossel  to  the  hexoses  of  the  carbohy- 
drate group  is  certainly  very  ingenious.  Usually  on  hydration  the 
^Zeits.  physiol.  Chem.,  25,168,  173. 


432  CHEMISTRY  OF  THE  LEUCOMAINS.. 

carbohydrates  yield  the  characteristic  hexose  unit.  On  similar  treat- 
ment the  protamins,  and  to  less  extent  the  more  complex  proteids, 
may  be  considered  as  yielding  the  equally  characteristic  hexon  unit. 
In  the  case  of  sturin  Kossel  and  Kutscher  ^  have  rendered  it  prob- 
able that  a  molecule  of  this  protamin  breaks  up  into  four  molecules 
of  arginin  and  one  each  of  histidin  and  lysin. 

In  view  of  the  marked  germicidal  properties  possessed  by  nucleinic 
acid  it  is  of  great  interest  to  note  that  the  protamins  are  likewise 
destructive  to  bacteria.  This  fact  has  been  shown  by  H.  Kossel  ^  who 
tested  the  action  of  sturin  and  of  its  carbonate  upon  a  number  of 
germs.  The  most  noticeable  effect  was  obtained  with  the  cholera 
vibrio.  These,  in  water  suspension,  were  killed  within  five  minutes 
by  sturin  carbonate  in  dilutions  as  high  as  1  to  10,000.  Even  a  1 
to  50,000  solution  exerted  a  marked  action,  though  the  time  required 
was  several  hours.  Typhoid  bacilli  and  staphylococci  were  obviously 
not  destroyed  as  readily,  but  a  marked  diminution  was  observed  when 
the  protamin  was  allowed  to  act  for  twenty-four  hours.  Spores  of 
anthrax  however  were  not  affected.  Solutions  of  protamin  carbonate 
in  ox-serum  in  a  strength  of  1  to  5,000  destroyed  anthrax  bacilli  within 
four  hours.  On  the  other  hand  cholera  and  typhoid  bacilli,  though 
greatly  diminished  in  numbers,  were  still  present  in  viable  condition 
at  the  end  of  that  time. 

It  is  evident  from  the  above  quoted  experiments  that  the  animal 
cell  possesses  germicidal  substances  other  than  nucleinic  acid.  It 
should  be  remembered,  however,  that  the  protamins  are  by  no  means 
widely  distributed ;  on  the  contrary  they  appear  to  be  restricted  to 
the  spermatozoa  of  certain  animals. 

Another  fact  brought  out  by  H.  Kossel  is  the  marked  poisonous 
action  of  protamins  and  of  histon.  Injected  subcutaneously  the  pro- 
tamins produce  in  guinea-pigs  severe  local  inflammation  which  brings 
on  extensive  exudate  and  finally  pronounced  necrosis.  When  injected 
intraperitoneally  or  intravenously  both  substances  cause  death  in  a 
few  minutes.  The  fact  that  histon  possessed  toxic  properties  was 
first  pointed  out  by  Novy.^  When  thymus  histon  was  injected  sub- 
cutaneously into  guinea-pigs  a  slight  temporary  depression  of  tem- 
perature followed.  This  was  followed  in  a  few  hours  by  an  increase 
of  1  to  1.5°,  which  would  persist  for  two  or  more  days.  Necrosis 
at  the  point  of  inoculation  was  frequently  observed.  In  rabbits  in- 
travenous injections  produced  exceedingly  rapid  effects,  death  often 
resulting  in  a  few  minutes. 

Since  then  the  toxic  action  of  histon  has  been  recognized  by  Kos- 
sel and  by  Thompson.  The  fall  in  blood  pressure,  the  change  in 
respiratory  movements,  the  retarding  effect  upon  coagulation  and  the 

^Zeits.  physiol.  Chem.,  31,  185  ;  25,  184. 
2  Zeits.  f.  Hygiene,  27,  36,  1898. 
''Journ.  Exp.  Med..,  1,  709,  1896. 


PROTAMINS  AND  HISTONS.  433 

agglutination  of  corpuscles  together  with  the  decrease  in  leucocytes 
is  no  wise  different  from  that  of  the  protamins.  This  interesting 
fact  is  readily  explainable  when  it  is  remembered  that  the  histons  are 
in  all  probability  compounds  of  protamins  and  proteids.  According 
to  Bang'  the  nucleoproteid  from  the  pancreas  and  its  cleavage  product 
guanylic  acid  possess  a  physiological  action  not  unlike  that  of  histon 
and  protamin. 

The  physiological  action  of  protamins  has  been  the  subject  of  a  care- 
ful study  by  Thompson^  who  showed  that  clupein  and  salmin,  which 
are  probably  identical,  and  scombrin  show  no  difference  in  their 
effects  whereas  the  more  complex  sturin  is  somewhat  less  poisonous. 
A  marked  and  relatively  rapid  fall  in  blood  pressure  was  observed 
shortly  after  the  injection  of  the  protamins.  In  dogs  death  rapidly 
followed  if  the  dose  exceeded  0,15-0.18  g.  of  clupein  per  10  kilo- 
grams of  body  weight.  The  limit  in  the  case  of  sturin  was  about 
0.20-0.25  g.  With  non-fatal  doses  the  blood  pressure  returns  to 
the  normal  in  about  half  an  hour  and  a  subsequent  injection  of  an 
otherwise  fatal  dose  is  tolerated.  According  to  Thompson  the  pro- 
tamins, like  the  albumoses,  act  directly  upon  the  walls  of  the  blood- 
vessels. The  effect  upon  the  respiration  is  extremely  marked  and 
when  death  occurs  it  is  due  to  paralysis  of  the  respiratory  move- 
ments. The  drop  in  the  blood  pressure  is  accompanied  by  an  in- 
crease in  the  depth  and  frequency  of  both  the  abdominal  and  thoracic 
respiratory  movements.  During  the  period  of  minimum  blood 
pressure  the  respiratory  movements  cease.  With  the  rise  in  pressure 
the  abdominal  respiration  is  resumed  but  the  thoracic  movements  are 
held  in  check  as  long  as  the  poison  continues  to  act.  The  effect  upon 
the  coagulation  of  blood  was  also  shown  to  be  marked.  While  a 
single  injection  shows  but  little  effect,  two  or  three  injections  delay 
the  coagulation  for  many  hours.  A  retardation  was  also  observed 
when  the  blood  was  added  to  protamin  in  a  test-tube.  It  is  worthy 
of  note  that  the  blood  became  more  transparent,  and  granular.  The 
corpuscles  thus  agglutinated  settle  rapidly,  leaving  a  clear  supernatant 
plasma.  In  this  respect  the  protamins  behave  like  albumose,  histon, 
venoms,  ricin,  guanylic  acid  and  other  agglutinants.  The  number  of 
circulating  leucocytes  was  appreciably  decreased. 

From  the  above  it  is  evident  that  the  protamins  and  histons  are 
active  poisons.  The  toxicity  is  connected  with  the  protamin  mole- 
cule as  a  whole  inasmuch  as  it  disappears  completely  on  cleavage. 
Thus,  the  very  earliest  hydrolytic  products,  the  proton,  or  pepton  of 
the  protamins,  exerts  but  very  little  effect  in  0.5  g.  doses,  and  the 
final  products  of  cleavage,  the  hexon  bases,  according  to  Thomson 
show  no  action  whatsoever  upon  the  blood  pressure  or  upon  the 
respiration.     Arginin  and  histidin,  like  antipepton,  hasten  the  coag- 

1  Zeits.  physiol.  Chem.,  32,  201, 1901. 
^Zeits.  physiol.  Chem.,  29,  1,  1900. 
28 


434  CHEMISTRY  OF  THE  LEUCOMAINS. 

ulation  of  blood.  The  inertness  of  arginin,  it  may  be  added,  was 
first  pointed  out  by  Schraiedeberg^  in  1896.  Matthews  has  shown 
that  arginin  in  large  amount  exerts  a  distinct  retarding  effect  upon 
tryptic  digestion. 

It  has  already  been  pointed  out  that  the  protamins  precipitate 
solutions  of  albumose  or  pepton  and  give  rise  to  histon,  or  at  all 
events  to  histon-like  bodies.  Inasmuch  as  the  natural  histons  show 
many  points  of  resemblance  to  the  protamins  it  is  quite  probable 
that  these  substances  represent  the  next  higher  group  of  proteids. 
A  brief  consideration  of  the  histons  in  this  connection  is  therefore 
by  no  means  out  of  place. 

The  first  representative  of  this  group  was  discovered  in  1884  by 
Kossel.^  Inasmuch  as  Miescher  had  previously  shown  that  the 
spermatozoa  of  the  salmon  contained  a  salt-like  combination  of  nu- 
clein  and  protamin,  the  thought  suggested  itself  that  a  similar  com- 
pound of  nuclein  and  a  basic  body  might  be  present  in  the  red 
corpuscles  of  the  blood  of  geese.  On  extracting  the  well  washed 
stroma,  perfectly  free  from  hemoglobin,  by  means  of  dilute  hydro- 
chloric acid  he  obtained  a  pepton-like  body  to  which  he  gave  the 
name  histon.  Ten  years  later  Lilienfeld'  showed  that  the  nucleo- 
histon  from  the  leucocytes  of  the  thymus  gland  on  treatment  with  dilute 
hydrochloric  acid  was  split  up  into  nuclein  and  histon.  The  latter, 
although  coagulable  by  heat,  he  regarded  as  identical  with  the  histon 
of  Kossel.  Very  soon  thereafter  histon  was  reported  to  be  present 
in  febrile  urine  (Krehl  and  Matthes*)  and  in  leukemic  urine  (Kolisch 
and  Burian^). 

An  examination  of  the  spermatozoa  of  the  sea-urchin  for  protamin 
by  Mathews  showed  that**  these  cells  contained  a  compound  of  nu- 
clein and  of  a  histon-like  body  to  which  he  gave  the  name  arbcudn. 
This  interesting  observation  is  substantiated  by  that  of  Miescher/ 
who  first  pointed  out  that  the  salmon  spermatozoa  if  immature  did 
not  contain  protamin,  but  instead  a  histon-like  substance — albumi- 
nose.  A  similar  condition  was  observed  by  Bang  ^  in  connection 
with  mackerel  spermatozoa.  These,  when  mature,  as  already  pointed 
out,  contain  the  protamin  scombrin,  but  the  immature  spermatozoa 
do  not  contain  the  substance,  but  instead  a  histon,  which  Bang 
designates  as  scombron.  Shortly  thereafter  Kossel  and  Kutscher® 
showed  that  the  same  was  true  for  the  mature  spermatozoa  of  the  cod 
(Gadus  morrhua).     Ehrstrom  ^^  arrived  at  a  similar  result  with  the 

»  Berichte,  29,  355. 

^ Zeits.  physioL  Chem.,  8,  511. 

^ Zeits.  physiol.  Chem.,  18,  482,  1894. 

*Archiv.  exp.  Path.  u.  Pharm.,  36,  441,  1895. 

^Zeits.  klin.  Med.,  29,  374,  1896. 

<^ZeiU  phydol.  Chem.,  23,  399,  1897. 

">  Archiv.*ezp.  Path.  u.  Pharm.,  37- 

^Zeits.  phydol.  Chem.,  27,  466,  1899. 

«Zeite.  physiol.  Chem.,  31,  191,  1900. 

i"Zeii8.  phydol.  Chem.,  32,  350,  1901. 


PBOTAMINS  AND  HISTONS.  435 

mature  spermatozoa  of  the  Lota  vulgaris,  a  fish  closely  related  to 
the  former. 

It  is  evident  from  these  investigations  that  while  the  mature 
spermatozoa  of  some  fish  contain  a  salt-like  combination  of  nucleinic 
acid  and  a  protamin,  the  same  spermatozoa  in  the  immature  condi- 
tion contain  a  similar  compound  in  which,  however,  the  protamin  is 
replaced  by  histon.  Again,  in  some  fish  as  the  cod  tribe  prota- 
min is  absent  even  from  the  mature  cells  and  its  place  is  taken 
by  histon.  Our  knowledge  of  the  composition  of  the  sperma- 
tozoa of  higher  animals  is  extremely  deficient.  According  to 
Mathews  the  spermatic  cells  of  the  steer  and  boar  contain  neither 
histon  nor  protamin,  while  according  to  Bang  the  liver,  kidney,  pan- 
creas and  testes  contain  no  histon.  At  least  no  such  body  could  be 
extracted  from  these  organs  by  dilute  acid.  On  the  other  hand,  the 
researches  of  Schulz  ^  show  that  the  proteid  constituent  of  hemo- 
globin, globin,  is  a  histon.  In  this  instance  the  histon  occurs  in 
combination  with  the  acid  hematin,  while  in  all  other  instances  it  is 
combined  with  nucleinic  acid.  Thus  far,  then,  histon  bodies  have 
been  shown  to  be  present  in  the  stroma  of  red  blood  cells  of 
geese,  in  hemoglobin,  in  the  leucocytes  of  the  thymus  gland,  usually 
in  the  immature  and  at  times  in  mature  spermatozoa  of  certain  ani- 
mals. 

Obviously,  as  in  the  case  of  the  protamins,  we  have  to  deal  with 
a  group  of  histons  and  not  with  a  single  individual.  With  this  fact 
in  mind  it  becomes  easy  to  reconcile  the  divergent  statements  which 
have  been  made  by  different  investigators  regarding  the  chemical 
reactions  of  these  substances.  Like  the  protamins,  the  histons  possess 
marked  basic  properties  and  on  decomposition  with  dilute  acid  they 
yield  the  hexon  bases.  The  amount  of  bases  is  by  no  means  as  large 
as  that  obtained  from  the  protamins,  but  at  the  same  time  it  is  much 
greater  than  that  from  other  proteids.  Looked  at  from  the  standpoint 
of  the  amount  of  hexon  bases  contained  in  the  molecule  the  prota- 
mins occupy  the  first,  the  histons  the  second  and  the  gelatins  the 
third  place.  The  thymus  histon,  according  to  Kossel,  yields  about 
40  per  cent,  of  its  nitrogen  in  the  form  of  hexon  bases,  while  ac- 
cording to  Lawrow  ^  these  bases  make  up  about  25  per  cent,  of  its 
weight.  The  hemoglobin  of  the  horse,  on  cleavage  with  hydro- 
chloric acid  and  tin,  yields,  according  to  the  same  observer,*  three 
hexon  bases  and  the  amount  corresponds  to  about  20  per  cent,  of  the 
globin  present.  The  Lota  histon  gave  Ehrstrom  12  per  cent,  ar- 
ginin,  3.17  per  cent,  lysin,  2.85  per  cent,  histidinand  0.66  per  cent, 
ammonia.  Analyses  of  gadus  and  thymus  histons  are  given  in  the 
table  on  p.  426. 

The  histons  yield  several  characteristic  reactions.     Neutral  solu- 

^Zeits.  physiol.  Chem.,  24,  449,  1898. 
^Zeits.  phyniol.  Chem.,  28,  390,  1899. 
^Berichte,  34,  101,  1901. 


436  CHEMISTRY  OF  THE  LEUCOMAINS. 

tions,  free  from  salts  of  ammonia,  are  precipitated  by  ammonia,  but 
the  precipitate  redissolves  in  excess  of  the  reagent.  The  scombron 
precipitate  however  does  not  redissolve.  In  the  presence  of  ammonia 
salts  the  precipitate  induced  by  ammonia  is  permanent.  As  pointed 
out  by  Bang  the  acid  albuminates  and  vitellin  are  likewise  precipitated 
by  ammonia.  The  fixed  alkalis  also  precipitate  histon.  While  pure 
solutions  of  histon  are  not  coagulated  on  boiling  this  does  occur  in 
the  presence  of  a  small  amount  of  salt.  As  Kossel  first  showed 
histon  may  be  precipitated  from  acid  solution  by  saturation  with 
sodium  chlorid  or  other  salts.  With  nitric  acid  the  histons  give 
the  albumose  reaction ;  likewise  the  xanthoproteic  reaction.  The 
biuret  test  is  given  by  all.  With  Millon's  reagent  the  reaction  is 
slight  but  distinct.  Salts  of  mercury  precipitate  some  histons,  but 
not  others.  The  most  important  reaction  is  based  upon  the  fact  that 
while  ordinary  proteids  are  precipitated  by  certain  alkaloidal  reagents 
in  acid  but  not  in  neutral  solution,  the  histons  like  protamins,  be- 
cause of  their  pronounced  basic  character,  are  precipitated  by  these 
reagents  from  neutral  as  well  as  acid  solutions.  Sodium  ferrocy- 
anid,  picrate,  phosphotungstate  and  phosphomolybdate  can  be 
thus  used.  Again,  like  the  protamins,  histon  precipitates  proteids,  ■ 
provided  free  alkali  is  not  present  in  excess.  This  property  is  like- 
wise seen  in  the  marked  agglutinating  action  which  histon  exerts  on 
bacteria.  On  digestion  with  pepsin  thymus  histon  is  split  up  and  a 
protamin-like  body  results  (Bang). 

The  physiological  action  of  histon  is  very  similar  to  that  of  prota- 
min  (p.  432).  The  action  of  thymus  histon  on  toxins  and  on  bac- 
teria with  special  reference  to  immunity  has  been  studied  by  Novy.^ 

Histon  may  be  prepared  direct  from  the  thymus  gland.^  For  this 
purpose  the  gland  is  finely  divided  and  extracted  with  water.  To 
the  aqueous  solution  hydrochloric  acid  is  added  to  0.8  per  cent,  con- 
centration. The  resultant  precipitate  is  removed  by  centrifugation 
and  from  the  clear  filtrate  the  histon  is  thrown  down  by  addition  of 
ammonia.  The  precipitate  is  washed  with  ammoniacal  water,  then 
with  alcohol  and  finally  with  ether. 

According  to  Fleroff^  the  thymus  gland  on  extraction  with  dilute 
sulphuric  acid,  as  in  the  method  for  protamins,  yields  histon  and 
para-histon.  The  latter  is  soluble  in  ammonia  and  water  and  is  thus 
separated  from  histon.  For  the  separation  of  histon  from  sperma- 
tozoa, see  Kossel  {Z.  P.  C,  31,  192);  from  hemoglobin,  see  Schulz 
(Z  P.  C,  24,  456). 

Arginin,  CgHj^NPg  • — The  study  of  the  constitution  of  this  base 
has  revealed  several  important  facts  which  enable  us  to  explain  sat- 
isfactorily a  number  of  vital  reactions.     The  observation  of  Drech- 

^Journ.  Exp.  Med.,  1,  709,  1896. 
'Zeits.  phydol.  Chem.,  31,  189  ;  28,  388. 
^ Zeita.  physiol.  Chem.,  28,  307. 


ARGININ.  437 

sel  in  1890  that  lysatinin  (p.  425)  on  boiling  with  baryta  gave  urea 
led  Schulze  and  Likiernik  to  make  a  similar  experiment  with  arginin. 
They  also  obtained  urea.  As  already  pointed  out  lysatinin  was  even- 
tually shown  to  be  a  mixture  of  arginin  and  lysin.  This  was  the 
first  time  that  urea  was  successfully  prepared  by  the  mere  hydration 
of  a  cleavage  product  of  proteids.  It  indicated  that  arginin  was 
without  doubt  a  guanidin  derivative. 

Subsequently  Schulze  and  Winterstein  ^  demonstrated  that  the 
cleavage  of  arginin  by  means  of  baryta  or  other  alkali  yielded  urea 
and  ornithin,  this  change  being  represented  by  the  equation  : 

CeH.AO^  +  H,0  =  CO(NH,),  +  C,H,,NA. 

Arginin.  Ornithin. 

Ornithin  had  been  demonstrated  by  Jaffe,  twenty  years  before,  to 
be  an  intermediate  waste  product  in  the  metabolism  in  chicken. 
When  benzoic  acid  is  introduced  into  the  body  of  mammals,  as  is 
well  known,  it  unites  with  glococoll  and  is  eliminated  as  hippuric 
acid.  In  birds  it  combines  with  ornithin  and  is  excreted  as  orni- 
thuric  acid,  Cj9H2oN20^ .  The  latter  was  shown  by  Jaffe  to  be  a 
dibenzoyl  ornithin  and  the  base  itself  he  regarded  as  a  diamido 
valerianic  acid  although  at  the  time  no  proof  of  this  could  be 
adduced.  On  treatment  with  nitrous  acid  Schulze  and  Winterstein 
demonstrated  the  presence  of  two  amido  groups  in  ornithin.  This 
fact  and  the  formation  of  urea  led  them  to  ascribe  to  arginin  the 
following  structure  which  subsequent  investigations  proved  to  be 
correct. 

NH, 


NH 
ifH.CHj.CH,.CH2.CH(NHj).C00H. 

It  is  evident  from  this  structure  that  arginin  is  closely  related  to 
creatin. 

5fH, 


.NH 
N(CH3).CHj.C00H. 

Analogous  to  the  synthesis  of  creatin  from  cyanamid  and  sarkosin, 
it  is  possible  to  effect  the  synthesis  of  arginin,  as  Schulze  and  Win- 
terstein ^  have  done,  by  the  action  of  cyanamid  on  ornithin.  These 
investigators  supplied  additional  evidence  in  favor  of  the  correctness 
of  the  structure  above  given  by  converting  ornithin  by  dry  distilla- 
tion and  hence  closing  the  chain,  into  a  pyrrolidin-like  body. 

In  1889  Udranszky  and  Baumann^  pointed  out  the  close  relation 

1  Zeits.  physiol.  Chem.,  26,  1,  1898  ;  34,  144. 

^Berichte,  32,  3191  (1899)  ;  Zeits.  physiol.  Chem.,  34,  128. 

^ Zeits.  physiol.  Chem.,  13,  590. 


438  CHEMISTRY  OF  THE  LEUCOMAINS.. 

between  ornithin  and  the  ptomain  putrescin  and  the  possibility  of 
the  latter  being  formed  by  splitting  off  carbonic  acid.  Although 
they  had  in  mind  experiments  in  this  direction  yet  nothing  was 
done  until  Ellinger^  effected  the  transformation.  On  decomposing 
ornithin  by  the  aid  of  bacteria,  especially  in  the  absence  of  oxygen, 
he  obtained  putrescin  and  thus  not  only  furnished  a  correct  explana- 
tion of  the  origin  of  this  ptomam  but  also  confirmed  the  position  of 
the  amido  groups  in  ornithin  and  hence  in  arginin.  The  cleavage  of 
proteids  by  bacteria  therefore  can  be  traced  through  arginin  and 
ornithin  to  putrescin.  The  structural  relation  of  these  bodies  can 
be  seen  from  the  equation  : 

NHa.CH2.CH2.CH,.CH(NH2).COOH  =  NHa.CHj.CHj.CHa.CHj.NH^  +  COj. 
Ornithin.  Putrescin. 

From  this  it  is  evident  that  ornithin  is  a-d  diamido  valerianic 
acid  and  is  the  next  lower  homologue  of  lysin  (see  p.  444).  This 
diamido  valerianic  acid  in  the  inactive  or  racemic  form  has  been 
recently  synthesized  by  Fischer.^  Like  ornithin  it  has  a  strong 
alkaline  reaction  and  gives  a  white  precipitate  with  mercuric 
chlorid ;  with  phosphotungstic  acid  it  yields  a  heavy  crystalline  pre- 
cipitate which  can  be  recrystallized  from  hot  water  in  colorless 
needles.  With  benzoyl  chlorid  it  forms  monobenzoyl  and  di-benzoyl 
derivatives  corresponding  to  those  of  ornithin.  While  Fischer  found 
the  natural  ornithuric  acid  to  be  dextro-rotatory,  the  synthetic  one 
was  inactive,  otherwise  the  two  showed  the  greatest  resemblance. 
The  synthetic  ornithuric  acid  melts  at  184-185°  whereas  the  natural 
acid  melts  at  182°  (Jaff6)  ;  184°  (Schulze  and  Winterstein).  Both 
yield  the  characteristic  calcium  salt.  The  rf-amido  valerianic  which 
Salkowski  isolated  from  putrefying  proteids  in  all  likelihood  is 
derived  from  the  former. 

On  oxidation  with  potassium  permanganate  arginin  is  destroyed 
but  by  employing  the  barium  salt  Benech  and  Kutscher^  succeeded 
in  oxidizing  the  base  with  the  formation  of  guanidin,  thus  demon- 
strating positively  the  presence  of  this  group  in  the  arginin  mole- 
cule. Just  as  creatin  is  a  methyl  guanidin  acetic  acid  so  arginin 
becomes  guanidin-a-amido  valerianic  acid.  On  further  examination 
of  the  oxidation  products  Kutscher*  obtained  /--guanidin  butyric 
acid  and  also  succinic  acid.  The  oxidation  of  arginin  can  be  shown 
as  follows : 

?H,  NH, 

.NH  C.NH 

NH.CHj.CH2.CH3.CH(NH2).COOH+Oj=ls^H.CH2.CH,.CH,.COOH+NH3-f-CO.,. 
Arginin.  Y-Gdanidin-butyric  acid. 

^Berichle,  31,  3183  (1898)  ;  Zeits.  physiol.  Chem.,  29,  334. 

^Berichte,  34,  454  (1901). 

'^  Zeits.  physwl.  Chem.,  32,  278. 

*  Zeits.  physiol.  Chem.,  32,413. 


ARGININ.  439 


I 


NH  C.NH 


NH.CH,.CH,.CH,.COOH  +  O,  =  NHj  +  COOH.CHj.CH,.COOH. 

GuANiDiK.  Succinic  Acid. 

The  demonstration  of  the  formation  of  succinic  acid  from  arginin 
explains  the  presence  of  this  acid  in  fermentation  and  putrefaction. 
Indeed  it  will  be  seen  that  the  study  of  the  cleavage  products  of 
proteids  and  more  directly  of  arginin  have  given  us  an  entirely  dif- 
ferent view  of  the  origin  of  urea,  guanidin,  putrescin  and  of  suc- 
cinic and  araido  valerianic  acids,  whether  formed  within  the  body, 
or  by  the  action  of  bacteria  or  of  chemicals  on  proteids  outside  of 
the  body. 

Arginin  exists  in  both  the  active  and  inactive  form.  According 
to  Kutscher^  all  proteids  except  fibrin  yield  the  dextro-rotatory 
base.  It  should  be  noted  in  this  connection  that  fibrin  is  the  only 
proteid  examined  which  has  been  submitted  to  tryptic  digestion  ;  all 
others  have  been  hydrolyzed  by  acids  and  it  is  quite  probable  that  if 
treated  with  trypsin  they  will  also  yield  inactive  arginin.  More- 
over, auto-digestion  of  the  pancreas,  if  short  in  duration,  may  yield 
also  the  inactive  form.  From  Balke's  antipepton  obtained  by  the 
pancreatic  digestion  of  fibrin  he  isolated  both  forms  whereas  in  auto- 
digestion  of  the  pancreas  only  the  active  arginin  was  found.  It 
would  seem  from  this  as  if  the  fibrin  either  contained  only  inactive 
arginin  or  else  it  contains  a  levo-form  which  racemizes  with  the 
dextro-arginin  derived  from  the  pancreas  proper.  The  dextro- 
arginin  on  heating  with  sulphuric  acid,  and  its  nitrate  when  ex- 
posed to  dry-heat  of  210°,  yields  the  inactive  form.  On  account  of 
slight  differences  Gulewitsch  believed  that  the  arginin  derived  from 
plants  was  distinct  from  that  of  animal  proteids  but  this  view  has 
been  shown  by  Schulze  ^  to  be  incorrect.  The  active  and  inactive 
arginins  are  the  only  two  kinds  known  at  present. 

The  free  base  can  be  prepared  from  the  acid  arginin  silver  nitrate 
by  changing  this  to  arginin  silver  which  in  turn  can  be  decomposed 
with  hydrogen  sulphid.  It  crystallizes  in  rosette-like  masses  of 
plates  or  thin  prisms  which  melt  at  207-207.5°  (corr.).  It  is  odor- 
less but  has  a  slightly  bitter  taste ;  is  strongly  alkaline  and  takes 
up  carbonic  acid.  From  solutions  of  the  salts  of  heavy  metals  it 
precipitates  the  oxids  and  from  ammonium  salts  it  liberates  ammo- 
nia. It  is  easily  soluble  in  water  but  is  almost  insoluble  in  boiling 
alcohol.  Like  ammonium  salts  and  amido  compounds  it  takes  up 
iodin  and  yields  iodic  acid.      (Schmidt,  Z.  P.  C,  34,  64.)     Sodium 

>  Zeits.  physiol.  Chem.,  32,  476  ;  26,  114  ;  28,  90. 
'^Zeits.  physiol.  Chem.,  27,  178,  368  ;  29,  329. 


440  CHEMISTRY  OF  THE  LEUCOMAINS. 

hypobromate  sets  free  about  one-third  of  its  nitrogen.  The  salts 
of  arginin  have  been  especially  studied  by  Gulewitsch.^ 

The  neutral  chlorid,  CgHj^N^Og.HCl  +  H2O,  is  easily  soluble  in 
water  from  which  it  can  be  crystallized  on  concentration  or  on  addi- 
tion of  alcohol  or  ether.  It  is  less  soluble  in  hot  than  in  cold 
85  per  cent,  alcohol.  It  forms  rosette-like  masses  of  plates.  The 
water  of  crystallization  may  be  driven  off  at  100°  (Hedin).  More- 
over, the  chlorid  may  crystallize  from  water  without  taking  up  water 
of  crystallization  (Schulze^).  Lawrow  obtained  a  chlorid  containing 
one-half  molecule  of  water.  The  anhydrous  salt  sinters  at  208°, 
gives  off  gas  and  melts  at  209°  or  higher.  When  heated  with 
concentrated  hydrochloric  acid  ammonia  is  split  off,  but  only  at  high 
temperatures  — 180°— 200°.  It  is  strongly  dextro-rotatory;  the 
rotation  is  increased  by  acid  and  decreased  by  alkali.  Hedin^  was 
unable  to  obtain  a  crystalline  acid  chlorid. 

The  neutral  nitrate,  CgHj^N^02.HN03  -f-  ^HjO,  results  on  decom- 
position of  the  basic  silver  or  copper  nitrate  salts.  It  can  be  read- 
ily crystallized  from  water  or  from  hot  dilute  alcohol,  forming 
opaque  chalk-like  masses  composed  ot  microscopic  needles  which  tend 
to  effloresce.  The  salt  is  somewhat  hygroscopic ;  when  dry  it  melts 
imperfectly  at  about  175°.  At  15°  it  is  soluble  in  about  two  parts 
of  water.  It  is  very  easily  soluble  in  hot,  difficultly  in  cold  85 
per  cent,  alcohol.  The  salt  is  dextro-rotatory  and  the  rotation  is 
markedly  affected  by  the  presence  of  free  acid.  It  dissolves  copper 
hydrate,  forming  the  double  salt. 

The  neutral  nitrate  of  the  inactive  arginin  which  Kutscher  iso- 
lated from  antipepton  (pp.  424,  439)  forms  small,  glistening,  trans- 
parent four-sided  prisms  or  plates,  which  often  unite  in  groups  and 
do  not  contain  water  of  crystallization.  It  is  more  difficultly  soluble 
than  the  dextro-salt  (1  to  17.25  water  at  20°;  1  to  21.7  parts  of 
water  at  12°)."  It  sinters  at  206°  and  melts  at  211°  with  gas 
formation. 

The  acid  nitrate,  CgHj^N^Oj.2HN03 ,  is  prepared  by  evaporating 
the  former  salt  with  excess  of  acid.  It  forms  long  colorless  needles 
or  masses  of  extremely  thin  elongated  plates.  The  melting  point  is 
144.5-145°. 

The  sulphate,  CgHj^N^Oj.HgSO^ ,  can  be  obtained  by  decomposing 
the  copper  sulphate  compound.  It  separates  from  saturated  alco- 
holic solution  as  an  oil  and  has  not  been  crystallized  (G.).  Hedin 
also  was  unsuccessful  in  crystallizing  the  sulphate.  It  is  hygro- 
scopic and  behaves  to  polarized  light  the  same  as  the  preceding  salts. 

'  Zeits.  physiol.  Chem.,  27,  178. 
'^Zeits.  physiol.  Chem.,  29,  331. 
^  Zeits.  physiol.  Chem.,  22,  157. 
*  Zeits.  TphysioL  Chem.,  32,  478. 


ARGININ.  441 

The  picrate,  C5Hj^N^02.CgH3N30^ ,  was  prepared  by  Schulze  and 
Steiger  (Z.  P.  C,  11,  43). 

Arginiu  copper  nitrate,  (CgHj^N^02)2  .Cu(N03)2  +  S^HjO,  was 
obtained  by  boiling  the  nitrate  with  copper  carbonate  or  hydrate. 
Hedin/  Lawrow,^  as  well  as  Schulze^  obtain  the  salt  with  only  three 
molecules  of  water,  possibly  because  of  loss  of  same  by  efflorescence. 
This  takes  place  slowly  when  the  salt  is  kept  in  a  desiccator.  When 
crystallized  from  hot  water  it  forms  roundish  aggregates  of  dark 
blue  needles  or  pointed  prisms.  Impure  solutions  are  difficult  to 
crystallize.  It  reacts  alkaline  and  is  soluble  in  95.5  parts  of  water 
at  13°.  It  melts  at  112°- 114°  but  when  dehydrated  it  melts 
with  decomposition  at  232°-  234°. 

The  copper  sulphate,  (CgHj^N^02)2.CuSO^+  SJIIgO,  was  obtained 
by  boiling  the  sulphate  with  cupric  hydrate.  It  melts  at  about  110° 
and  loses  all  the  water  of  crystallization  at  150°  and  decomposes  at 
170°.     The  dehydrated  salt  decomposes  at  235°-  238°. 

The  acid  silver  nitrate  compound,  CgHj^N^02.  IINO3  +  AgNOg , 
on  slow  evaporation  of  its  aqueous  solution  forms  long  colorless 
needle-shaped  prisms  usually  grouped  in  bundles.  From  hot  satu- 
rated solution  it  separates  in  opaque  stellate  masses  of  long,  very 
thin  needles.  Its  solution  which  is  very  slightly  acid  is  not  reduced 
on  heating.  It  is  soluble  in  7.27  parts  of  water  at  16° ;  is  insolu- 
ble in  alcohol  and  in  ether,  and  is  dextro-rotatory.  It  melts  with 
decomposition  at  176°- 183°. 

The  basic  silver  nitrate  compound,*  CgH^^N^Oj.AgNOj  -f  ^HgO, 
forms  rosette  or  wart-like  aggregates  of  short,  colorless,  transparent 
prisms.  Its  solution  reacts  alkaline  ;  is  easily  reduced  by  light  and 
by  heating.  At  16°  it  dissolves  in  88.7  parts  of  water  (81  parts, 
Hedin) ;  is  easily  soluble  in  hot  water.  It  is  insoluble  in  alcohol  and 
in  ether.  It  decomposes  at  164°.  This  salt  has  been  used  as  a 
means  of  separation  (Hedin). 

Arginin-silver,^  CgHj2Ag2N^02.Il20,  is  obtained  as  a  snow-white 
cheesy  precipitate  when  sodium  hydrate  is  added  carefully  to  the 
acid  silver  nitrate  compound.  It  is  usually  mixed  with  the  similar 
body  containing  three  atoms  of  silver.  The  compound  takes  up 
carbonic  acid ;  when  powdered  is  markedly  electric  and  darkens  on 
exposure  to  light.  The  moist  powder  when  heated  rapidly  becomes 
black.  It  is  very  difficultly  soluble  in  water  (1  in  28,571);  easily 
soluble  in  acids  and  in  ammonia. 

A  mercuric  chlorid  compound  forms  when  this  salt  is  added  to  a 
solution  of  arginin.  The  voluminous,  white,  amorphous  precipitate  is 
soluble  in  acids,  and  in  hot  water.     When  purified  it  becomes  very 

J  Zeits.  physiol.  Chem.,  20,  191. 

'Zeits.  physiol.  Chem.,  28,  392. 

'Zeits.  physiol.  Chem.,  11 ;  52  ;  29,  331  ;  33,  561  ;  34,  138. 

*  Zeits.  physiol.  Chem.,  20,  188  ;  21,  156  ;  27,  200. 

^ Zeits.  physiol.  Chem.,  22,  194  ;  27,  202. 


442  CHEMISTRY  OF  THE  LEUCOMAINS. 

difficultly  soluble  in  boiling  water.  It  melts  and  decomposes  at 
186°- 189°.  Potassium  mercuric  iodid  alone  gives  no  precipitate 
but  on  addition  of  sodium  hydrate  a  heavy  white  precipitate  forms. 
Potassium  bismuth  iodid  gives  a  red  precipitate.  The  picrate  melts 
at  205°. 

The  additibn  of  phosphotungstic  acid  to  solutions  of  the  nitrate 
produces  a  pulverulent  precipitate  which,  recrystallized  from  boiling 
water,  forms  very  small  prisms  or  needles.  It  is  soluble  in  large 
excess  of  the  reagent. 

Dibenzoyl  arginin,  CgH^2(^6^5-^^)2^4^2  j  ^^  obtained  readily, 
though  the  yield  is  small.  On  treatment  with  benzoyl  chlorid  a 
slight  sticky  precipitate  forms  and  from  the  filtrate  the  dibenzoyl 
compound  is  thrown  down  on  acidulation  and  is  separated  from  ben- 
zoic acid  by  means  of  ether.  From  boiling  water  it  crystallizes  in 
masses  of  very  fine  long  needles  or  rhombic  plates.  The  needle- 
shaped  crystals  are  often  branched,  or  stellate  and  show  split  ends. 
It  is  difficultly  soluble  in  boiling  water ;  more  readily  in  boiling  alco- 
hol ;  easily  in  alkali.  It  melts  with  slight  decomposition  at  217°- 
218°.     This  body  is  analogous  to  ornithuric  and  lysuric  acids. 

Histidin,  CgHgNgOg. — As  yet  nothing  definite  is  known  regarding 
the  constitution  of  this  base.  From  the  small  amount  of  hydrogen 
present  it  would  seem  that  the  structure  of  histidin  must  be  that  of 
a  closed  chain.  When  it  is  remembered  that  cadaverin  on  dry  dis- 
tillation yields  piperidin  (p.  273),  it  does  not  seem  improbable  but 
that  arginin,  the  parent  substance  of  cadaverin,  may  in  a  similar 
manner,  by  closing  the  chain  yield  histidin.  The  change  in  the  case 
of  cadaverin  is  represented  thus  : 


NH,.CH3.CH2.CH2.CH.2CH2.NH2    =  ^^  1       L^     +    NHj. 


H2 

c 

/\ 

H,(1       CH, 

CH3.CH2.CH2.CH.2CH2.NH2      : 

~  H„C       CH 

V 

H 

Cadaverin. 

PlPBRIDIN. 

The  transformation  of  arginin  into  histidin  would  in  a  similar  way 
be  represented  by  the  equation : 


NH 
NH.CHj.CHj.CHj.CH(NHj).COOH 

Arginin.  Histidin  (7) 


NH — 

C.NH 

1 

-CHCOOH 

in, 

CH, 

-6h, 

NH — 

+  NH3. 


If  the  above  change  is  correct  histidin  would  possess  the  formula — 


HISTIDIN.  443 

CgH^jNgOg .  The  analytical  results  correspond  about  as  closely  with 
the  theoretical  percentages  required  for  this  formula  as  with  CgHgNjO^.^ 
According  to  this  interpretation  of  histidin,  like  arginin,  it  should 
yield  on  cleavage  urea,  guanidin  and  d  amido  valerianic  acid.  The 
latter  has  been  repeatedly  found  among  the  cleavage  products  of  the 
protamins.^  The  conversion  of  creatin  into  creatinin  is  analogous  to 
that  shown  above  with  the  difference  however  that  water  and  not 
ammonia  is  given  off. 

If  the  above  view  of  the  origin  of  histidin  be  correct  it  will  be 
seen  that  the  hexon  bases  acquire  a  new  importance  in  so  far  as  they 
would  form  antecedents  of  the  pyrimidin  and  even  purin  bodies. 
The  nucleus  of  the  cell  therefore  would  transform  the  ready-made 
protoplasmic  bases  into  the  characteristic  nuclein  bases. 

Histidin  itself  forms  well  defined  rhombic  plates  or  needles.  It 
is  not  very  soluble  in  water  and  hence  can  be  crystallized  from  it 
direct  or  by  addition  of  alcohol.  The  solution  shows  faint  alkaline 
reaction  and  does  not  take  up  carbonic  acid  (Hedin).  It  is  insoluble 
in  alcohol  and  in  ether.  According  to  Kossel,^  the  free  base  is  levo- 
while  its  salts  are  dextro-rotatory ;  and  the  rotation  is  increased  by 
the  presence  of  free  acid.  Histidin  from  thymus  histon,  according 
to  Lawrow/  is  dextro-rotatory,  which  fact  considered  in  connection 
with  the  difference  observed  between  the  chlorid  of  histidin  from 
histon  and  from  other  sources,  may  be  taken  to  indicate  different 
histidins. 

The  free  base  is  usually  obtained  from  the  chlorid  by  decomposi- 
tion with  silver  sulphate.  Possibly  histidin  unites  with  either  of 
the  other  hexon  bases.  This  is  indicated  in  the  isolation  of  a  com- 
pound, CjgHggNyOg,  by  Kossel  and  Mathews,^  from  sturin  by  diges- 
tion with  trypsin.  This  compound  consists  probably  of  one  mole- 
cule of  histidin  and  two  molecules  of  lysin.  Sturin  itself  probably 
contains  the  three  bases  in  combination. 

The  monochlorid,"  CgHgN302.HCl  -|-  H^O,  is  obtained  on  decom- 
position of  the  histidin-silver  compound  with  hydrochloric  acid 
(Hedin)  and  crystallizes  in  fine,  large,  glassy  plates  which  do  not  lose 
water  of  crystallization  at  120°,  but  do  at  135°  (Hedin),  at  105° 
(Kossel).  It  melts  at  251°- 252°;  is  easily  soluble  in  water,  in- 
soluble in  alcohol  and  in  ether.  The  aqueous  solution  is  acid  in  re- 
action and  is  slightly  dextro-rotatory,  although  Hedin  first  consid- 
ered it  to  be  inactive.  The  chlorid  from  gelatin  and  from  silk  melts 
at  243°  (Wetzel). 

The  di-chlorid,^  CgHgN302.2HCl,  contains  no  water  of  crystalliza- 

^Zeits.  phydol.  Chem.,  22,  184,  194. 

"^Zeits.  physiol.  Chem.,  26,  590  ;  31. 

^Zeits.  physiol.  Chem.,  28,  382. 

* Zeits.  physiol.  Chem.,  28,  392;  31,  193. 

^Zeits.  phydol.  Chem.,  25,  193  ;  31,  185. 

^  Zeits.  physiol.  Chem. ,  22,  182,  192;  25,  192;  28,  387,  460. 

''Zeits.  physiol.  Chem.,  28,  383,  392  ;  31,  189,  193. 


444  CHEMISTRY  OF  THE  LEUCOMAINS. 

tion  which  in  the  above  appears  to  be  replaced  by  the  second  mole- 
cule of  acid  (Schwantke).  It  can  be  obtained  from  the  monochlorid  by 
repeated  treatment  with  concentrated  hydrochloric  acid  (Kutscher). 
On  slow  crystallization  it  forms  large  glassy  plates,  except  when 
the  salt  is  derived  from  histon,  in  which  case  it  crystallizes  slowly 
and  with  different  form.  Differences  in  solubility  have  also  been 
noted.  It  sinters  at  225°  and  finally  melts  at  231°- 233°  with 
evolution  of  ammonia.  The  latter  is  also  given  off  on  prolonged 
heating  at  140°. 

The  nitrate,  CgHgN302.2IIN03,  was  obtained  by  KosseP  in  crys- 
talline form,  although  Hedin  was  unsuccessful  in  doing  so  with  the 
nitrate  or  sulphate.  With  platinum  chlorid  and  silver  nitrate  crys- 
tallizable  double  salts  are  obtainable.  A  rather  uncertain  double 
salt  of  barium  chlorid  has  been  reported  (impure  dichlorid  ?). 

Mercuric  chlorid  precipitates  the  alkaline  carbonate  solution  of 
histidin  and  this  fact  was  utilized  at  one  time  by  Kossel^  as  a  means 
of  separation  from  arginin. 

Silver  nitrate  when  added  to  an  aqueous  solution  of  the  base  pro- 
duces no  precipitate,  but  on  addition  of  ammonia  a  voluminous  pre- 
cipitate forms  which  is  easily  soluble  in  excess  (Hedin).^  Its 
formula  is  CgIIyAg2N302  -f-  HgO.  Instead  of  ammonia  Kossel  em- 
ploys baryta,  which  on  careful  addition  throws  down  the  histidin 
compound  first  and,  subsequently  in  strong  excess,  precipitates  the 
arginin  compound.  These  two  bases  can  thus  be  easily  separated 
(see  p.  427). 

Lysin,  CgH^^NgOg. — The  relation  of  this  base  to  leucin,  CgH^gNOj, 
from  which  it  differs  by  an  NHg  group  led  Drechsel  *  to  regard  it  as 
a  di-amido  caproic  acid.  He  attempted,  though  unsuccessfully,  the 
conversion  of  lysin  into  cadaverin  and  even  pointed  out  its  possible 
transformation  into  pyridin.  Inasmuch  as  lysin  appeared  to  be  a 
homologue  of  ornithin,  C^^^P^,  Ellinger^  reasoned  that,  like  the 
latter,  it  should  yield  on  putrefaction  the  diamin  cadaverin,  the 
homologue  of  putrescin.  Experiments  with  lysin  derived  from 
casein  and  from  auto-digested  pancreas  gave  the  expected  cadaverin 
and  at  the  same  time  established  the  constitution  of  the  former. 
Lysin,  therefore,  is  a  —  £  di-amido  caproic  acid  and  is  the  next 
homologue  of  ornithin.  The  cleavage  into  cadaverin  can  be  repre- 
sented by  an  equation  similar  to  that  for  ornithin  (p.  438). 

NHj.CH,.CH,.CH,.CH,.CH(NH2).C00H  =  NHa.CH,.CH2.CH,.CH,.CHj.NHj  +  CO^ 
Lysin.  Cadavkbin. 

^Zeits.  physiol.  Chem.,  22,  195  ;  28,  383,  391. 

^Zeits.  physiol.  Chem.,  22,  182;  31,  171. 

^Zeils.  physiol.  Chem.,  22,  192  ;  31,  171  ;  25,  171  ;  28,  460. 

« Berichie,  25,  2456,  3504. 

^Zeits.  physiol.  Chem.,  29,  334,  1900. 


LYSIN.  445 

Since  lysin  is  a  homologue  of  ornithin  it  is  possible  that  like  the  latter 
it  exists  in  the  proteid  molecule  in  combination  with  an  urea  residue. 
In  other  words  it  may  have  a  parent  substance  which  bears  to  it  the 
same  relation  that  arginin  does  to  ornithin.  This  theoretical  body 
would  possess  the  formula  C^HjgN^Oj .  On  hydration  it  would  yield 
urea  and  lysin  in  the  same  way  that  arginin  yields  urea  and  ornithin. 
In  this  way  perhaps  may  be  explained  the  origin  of  the  ammonia 
which  is  formed  during  the  hydrolysis  of  proteids  (see  table,  p.  426). 
The  existence  of  the  ptomain  hexamethylene  diamin  may  be  taken  to 
indicate  a  still  higher  homologue.  The  relation  of  these  bodies  may 
be  indicated  thus  : 

Arginin,  CgHj^N^O,  =  Ornithin,  CsHj^NjOj  =  Putrescin,  O^HijN,. 

=  Lysin,        CgH^^NjOj  =  Cadaverin,  C5H,4Nj. 

=  Hexa-met. -diamin,  CgHijNj. 

Lysin  is  easily  soluble  in  water  and,  like  arginin  and  histidin,  it  is 
not  precipitated  on  saturation  with  ammonium  sulphate  but  is  pre- 
cipitated by  alcohol  and  by  phosphotungstic  acid.  Hence  the  pres- 
ence of  these  bases  in  antipepton  (Kutscher).  Unlike  the  other  two 
bases  lysin  is  not  precipitated  by  silver  nitrate  and  fixed  alkali,  and 
this  fact  Kossel  makes  use  of  to  effect  their  separation  (p.  428). 
The  free  base  can  be  obtained  best  from  the  sulphate.  On  heating 
it  gives  off  alkaline  vapors  (Drechsel,  Kruger).  On  fusion  with 
alkali  it  yields  acetic  and  propionic  acids  (Henderson). 

In  the  tryptic  digestion  of  sturin  Kossel  and  Mathews  obtained 
a  compound,  CjgHj^NyOj ,  which  crystallized  in  needles  and  was  evi- 
dently a  combination  of  two  molecules  of  lysin  with  one  of  histidin. 
The  lysatinin  (p.  425)  of  Drechsel  is  apparently  a  similar  combina- 
tion of  one  molecule  each  of  lysin  and  of  arginin. 

The  dichlorid,  CgHj^N202.2HCl,  can  be  prepared  from  the  picrate 
or  from  the  platinum  salt.  It  melts  at  217°  and  at  261°  it  decom- 
poses yielding  hydrochloric  acid,  carbon  monoxid,  water  and  a 
sublimate  of  ammonium  chlorid.  DrechseP  interpreted  this  reac- 
tion as  leading  to  amido  valeric  aldehyde  which  by  loss  of  water  should 
yield  tetrahydropyridin  but  this  transformation,  which  would  be 
analogous  to  the  change  of  cadaverin  into  piperidin,  has  not  as  yet 
been  realized.  According  to  Henderson  it  melts  at  192-193°; 
while  Lawrow^  states  that  it  begins  to  melt  at  194—195°  and  gives 
off  gas  at  200-202°.  It  is  not  changed  to  the  monochlorid  by  pro- 
longed heating  at  130°.  The  aqueous  solution  is  strongly  acid  and 
on  evaporation  with  the  alkali  it  forms  bases  possessing  a  slight 
coniin-like  odor  (Drechsel).  The  colorless  crystals  are  but  slightly 
soluble  in  alcohol ;  readily  soluble  in  methyl  alcohol,  and  readily 
form  from  aqueous  solution  on  concentration. 

The  salt  as  well  as  the  free  base  are  dextro-rotatory  and  the  devia- 

1  Berichte,  25,  2455,  3504. 

'^Zeits.  physiol.  Chem.,  28,  395  ;  29,  321. 


446  CHEMISTRY   OF  THE  LEUCOMAINS. 

tion  of  polarized  light  is  increased  by  acids  (Lawrow).  On  heating 
with  baryta  it  becomes  inactive  (Siegfrid,  Lawrow).  For  its  be- 
havior to  phenyl  isocyanate  see  ornithin.  Henderson^  examined 
lysin  from  six  different  sources  without  finding  any  difference. 

The  picrate,'  CgHj^N^O^-CgHgNgO^,  is  difficultly  soluble  and  is 
therefore  especially  useful  for  the  isolation  of  the  base  (p.  428).  It 
separates  in  large  crystals,  from  solutions  of  the  base  in  alcohol,  on 
the  addition  of  sodium  picrate.  It  is  soluble  in  185  parts  of  water 
(Lawrow). 

The  carbonate,  2CgHj^N202  +  COg ,  was  obtained  in  crystalline  form 
by  Drechsel  ^  and  was  regarded  as  analogous  to  ammonium  car- 
bamate. 

Phosphotungstic  acid  gives  a  precipitate  which,  when  recrystallized 
from  much  boiling  water,  forms  needles  (Kutscher*). 

Platinum  chlorid  precipitates  lysin  from  alcoholic  solution  as 
CgHj,N202.2HCl.PtCl,  +  C^HgO  (Drechsel).  It  forms  fine  yel- 
lowish red  prisms  from  which  the  alcohol  of  crystallization  can  be 
expelled  by  drying  at  130°.     It  melts  at  219°-220°   (Schulze^). 

Lysin  like  arginin,  forms  two  silver  salts.  The  compound 
CgHj^NgOj.AgNO,  is  more  soluble  than  the  corresponding  one  of 
arginin  and  has  been  used  as  a  means  of  separation  (Hedin  ^).  The 
acid  compound,  CgHj^N202.HN03' +  AgN03 ,  is  easily  soluble  and 
crystallizes  from  water  by  addition  of  alcohol  and  ether  in  beautiful 
needles. 

Mercuric  chlorid'''  does  not  precipitate  dilute  solutions  of  lysin 
and  arginin.  The  nitrate  by  itself  does  not  precipitate  lysin  or 
arginin  but  upon  the  addition  of  sodium  hydrate  these  bases  are 
thrown  down. 

With  benzoyl  chlorid^  lysin  forms  a  di-benzoyl  derivative — 
lysuric  acid  —  which  corresponds  to  ornithuric  and  hippuric  acids. 
This  compound  forms  acid  as  well  as  neutral  salts  which  have  been 
studied  by  Drechsel  and  by  Willdenow.  The  acid  barium  salt  crys- 
tallizes easily  and  is  useful  for  the  separation  and  purification  of 
lysin.  In  the  presence  of  acids,  lysuric  acid  is  soluble  in  ether  but 
not  in  petroleum  ether  which  therefore  can  be  used  for  its  separation 
(Lawrow  ^).     A  mono-benzoyl  derivative  has  also  been  prepared. 

Ornithin,  C5Hj2N202,  was  discovered  in  1877  by  Jaff6  who  ob- 
tained it  in  the  form  of  ornithuric  acid  from  the  urine  of  chicken 

^  Zeits.  physiol.  Chem.,  29,  323. 

'^Zeits.  physiol.  Chem.,  25,  180  ;  26,  586  ;  28,  398  ;  33,  553. 

3  Berichte,  25,  2455. 

*Zeits.  physiol.  Chem.,  28,  89. 

^Zeits.  phymd.  Chem.,  28,  469  ;  33,  561. 

^ Zeits.  physiol.  Chem.,  11,  300,  303. 

">  Zeih.  phydol.  Chem.,  21,  303;  25,  176. 

^Berichte,  28,  3189  ;  Zeits.  physiol.  Chem.,  25,  522 ;  26,  398  ;  33,  562. 

'  Zeits.  physiol.  Chem. ,  28,  585. 


ORNITHIN.  447 

after  administration  of  benzoic  acid.  This  acid  as  pointed  out  on 
p.  437  is  a  di-benzoyl  compound  of  ornithin  and  yields  the  base  on 
cleavage  with  concentrated  acid.  Ornithin  therefore  can  be  looked 
upon  as  an  intermediate  waste  product  (and  possibly  as  an  uric 
acid  antecedent)  in  the  chicken  just  as  glycocoll  is  such  in  other  ani- 
mals. The  base,  however,  is  by  no  means  peculiar  to  the  chicken, 
inasmuch  as  it  exists  preformed  in  arginin  which,  as  pointed  out 
(p.  426),  is  present  in  all  proteid  matter,  whether  of  animal  or  plant 
origin.  The  partial  synthesis  of  arginin  from  ornithin  and  cyanamid 
has  been  referred  to.     Ornithin  can  be  prepared  readily  from  arginin.^ 

The  belief  of  JaiF(§  that  ornithin  was  a  di-amido  valerianic  acid  has 
been  definitely  established  in  the  past  year  or  two.  The  cleavage  of 
arginin  into  urea  and  ornithin  and  the  decomposition  of  the  latter  by 
bacteria  into  putrescin  demonstrate  that  this  peculiar  base  is  a-d 
diamino  valerianic  acid  (p.  438). 

The  synthesis  of  a-d  diamino  valerianic  acid  has  only  recently 
been  effected  by  Fischer.^  It  yields  with  benzoyl  chlorid  a  di-ben- 
zoyl derivative,  CjgHjgNjO^,  which  is  strikingly  similar  to  ornithuric 
acid.  While  the  latter  melts  at  182°  (Jaflpg),  184°  (Schulze  and 
Winterstein),  the  synthetic  product  melts  at  184°- 185°.  The  syn- 
thetic and  natural  ornithuric  acids  yield  the  same  characteristic  cal- 
cium salt,  (CjgHjgN20^)2Ca.  They  both,  on  partial  cleavage,  form 
the  mono-benzoyl  derivative,  which  in  the  case  of  the  natural  base 
crystallizes  in  needles  (Jaff§),  whereas  the  synthetic  body  forms  plates. 

The  synthetic  ornithin  is  strongly  alkaline  in  reaction,  gives  a 
flocculent  white  precipitate  with  mercuric  chlorid  and  in  acid  solu- 
tion gives  with  phosphotungstic  acid  a  heavy  crystalline  precipitate 
which  easily  dissolves  on  heating  and  reappears  on  cooling  in  color- 
less needles.  The  only  real  difference  between  the  synthetic  and 
natural  ornithin  is  in  the  fact  that  the  latter  is  dextro-rotatory,  while 
the  former  is  inactive.  The  synthetic  ornithin  is  therefore  the  racemic 
form  of  the  natural  base  as  obtained  from  chicken  and  from  arginin. 
The  properties  of  the  base  and  of  several  of  its  salts  have  been  de- 
scribed by  Schulze  and  Winterstein.^  Like  lysin  it  unites  with 
phenyl  isocyanate  to  form  an  addition  product  which  with  hydro- 
chloric acid  yields  an  easily  crystallizable  hydantoin  (Herzog*). 

Ornithin  on  cleavage  by  bacteria  yields  putrescin  and  it  is  very 
probable  that  this  change  may  be  effected  by  chemical  means  and 
also  by  metabolism  within  the  body.  Indeed  Lawrow's^  studies 
upon  the  auto-digestion  of  pig's  stomachs  show  that  pepsin  may 
split  up  proteids  into  monamido  acids  and  the  diamins — cadaverin  and 
putrescin.     The  occurrence  of  these  bases  in  cystinuria  may  be  ac- 

'  Zelts.  physiol.  Chem.,  34,  525. 
^Berichte,  34,454  (1901). 
3  Zeits.  physiol.  Chem.,  34,  128. 
*Zeits.  physiol.  Chem.,  34,  525. 
^ Zeits.  physiol.  Chem.,  33,  312. 


448  CHEMISTRY  OF  THE  LEUCOMAINS. 

counted  for  by  abnormal  metabolism  rather  than  by  bacterial  activity, 
and  if  so,  it  may  be  possible  to  demonstrate  the  presence  of  the  ante- 
cedents ornithin  and  lysin. 

The  relation  of  lysin  to  cadaverin  and  of  the  latter  to  piperidin 
(p.  442),  suggests  a  similar  relation  between  ornithin,  putrescin  and 
pyrrolidin.  The  carboxyl  derivative  of  the  latter  has  been  obtained 
from  proteids  and  will  be  described  next. 

a-Pyrrolidin  carbonic  acid,  C5H9NO2,  is  a  product  ot  proteid 
cleavage  which  differs  from  all  others  in  that  it  has  a  nitrogen  con- 
taining ring.  It  undoubtedly  originates  from  and  is  related  to  the 
hexon  bases  arginin,  histidin  and  ornithin.  This  is  seen  in  the  fact 
that  Schulze  and  Winterstein  obtained  a  pyrrolidin-like  body  on  dry 
distillation  of  arginin  and  of  ornithin.  On  the  other  hand,  although 
this  base  results,  according  to  Fischer,  in  the  hydrolytic  decomposi- 
tion, with  hydrochloric  acid,  of  casein,  egg-albumin,  fibrin  and  gelatin 
(Levene),  it  does  not  form  when  arginin  or  ornithin  are  subjected  to 
the  same  method.  For  this  reason  Fischer '  is  inclined  to  consider 
it  as  a  primary  cleavage  product  of  proteids.  It  is  conceivable,  how- 
ever, that  this  base  may  originate  in  the  cleavage  of  histidin  or  its 
parent  substance  (p.  442)  under  conditions  similar  to  the  forma- 
tion of  uric  acid  out  of  hypoxanthin  or  out  of  thymus  gland  (p.  344), 
in  which  case  urea  would  be  split  off  and  the  chain  would  then  re- 
close  to  form  the  pyrrolidin  carbonic  acid. 

The  presumed  formation  of  this  base  from  ornithin  can  be  ex- 
pressed by  the  following  equation  : 

CH, — Y    2 
NH,.CHj.CH,.CHj.CH(NHa).COOH    =    CH„    CH.COOH -f  NH,. 

NH 

This  equation  should  be  compared  with  those  for  piperidin  and  his- 
tidin on  page  442.  It  is  noteworthy  that  this  acid  is  closely  related 
to  the  cleavage  products  of  atropin  and  cocain,  namely  tropic  and 
hygrinic  acids.  Moreover,  as  indicating  the  possibility  of  its  being 
a  secondary  product  of  proteid  cleavage  may  be  mentioned  the  fact 
that  a  methyl  1-4  di-amido  valerianic  acid  on  treatment  with  hydro- 
chloric acid  yields  a  methyl  pyrrolidin  carbonic  acid  (Wildstatter 
and  Ettlinger^). 

The  synthesis  of  a-pyrrolidin  carbonic  acid  has  been  effected  in- 
dependently by  "Wildstatter^  and  by  Fischer.*  As  in  the  case  of 
ornithin  the  synthetic  body  is  the  racemic  form  whereas  the  natural 
product  is  both  active  and  inactive.     Thus,  from  casein  Fischer  ob- 

^Zeits.  physiol.  Chem.,  33,  162,  163,  412. 
'Benchte,  35,620. 
'■'Berichte,  33,  1160. 
*Berichte,  34,454(1901). 


GERONTIN.  449 

tained  about  3.2  per  cent,  yield  of  the  acid,  and  of  this  about  two- 
thirds  was  levo-rotatory,  the  remainder  being  inactive.  On  heating 
with  baryta  under  pressure  the  active  form  is  changed  into  the  inac- 
tive. 

The  free  acid  melts  at  203-206°  (198°  Wildstatter)  with  decom- 
position and  gives  off  an  odor  of  pyrrolidin.  This  odor  is  also  noted 
during  the  evaporation  of  a  solution.  It  forms  flat  needles  which 
effloresce.  Owing  to  its  marked  solubility  in  water  and  in  alcohol 
it  was  overlooked  in  previous  studies  of  casein.  It  gives  with  phos- 
photungstic  acid  a  crystalline  precipitate  which  easily  dissolves  on 
boiling. 

The  active  form  unites  in  alkaline  solutions  with  phenyl  isocyanate 
to  form  a  cyanate,  the  anhydrid  of  which  readily  crystallizes  and 
melts  at  143°,  whereas  that  from  the  inactive  form  melts  at  118°. 

On  boiling  with  freshly  precipitated  copper  oxid  the  acid  forms 
the  easily  crystallizable  salt  (C5HgN02)2Cu  +  2H2O,  which  is  espe- 
cially useful  for  the  isolation  of  the  acid.  The  salts  of  both  active 
and  inactive  forms  possess  the  same  properties. 

The  ethyl  ester  boils  at  75°  -  76°  at  11  mm.  pressure.  It  was  by 
fractional  distillation  of  the  esters  of  the  monamino  acids  which  re- 
sult in  the  hydrolytic  cleavage  of  proteids  that  this  acid  was  isolated 
by  Fischer. 

Gerontin,  Cgllj^Ng,  is  a  base  which  was  isolated  by  Grandis  in 
1890.  It  has  been  repeatedly  observed  in  the  form  of  peculiar  crys- 
tals found  in  the  cell  nuclei  in  the  liver  and  kidneys,  particularly  of 
old  dogs.  The  free  base  is  an  isomer  of  cadaverin,  etc.,  and  re- 
sembles it  somewhat.  It  crystallizes  in  needles  which  are  readily 
soluble  in  water  and  alcohol ;  possesses  a  strongly  alkaline  reaction, 
and  yields  the  ordinary  alkaloidal  reactions. 

The  hydrochlorid  forms  prismatic  crystals,  which  are  deliquescent 
and  easily  soluble  in  alcohol. 

The  platinochlorid,  C^^^^.'lllCl.ViQ^,  is  soluble  in  water  and 
crystallizes  in  spindle-shaped  needles,  arranged  in  rosettes.  It  de- 
composes at  115°. 

The  gold  salt  forms  small  needles,  and  is  easily  soluble  in  water 
and  alcohol. 

It  combines  with  one  molecule  of  mercuric  chlorid  to  form  deli- 
quescent cubes  or  rectangular  prisms  containing  two  molecules  of 
water  of  crystallization.  It  decomposes  above  100°.  This  distin- 
guishes it  from  cadaverin,  which  combines  with  three  to  four  mole- 
cules of  mercuric  chlorid.  The  crystals  observed  in  the  liver  are 
probably  the  phosphate. 

The  new  base  also  yields  a  benzoyl  compound  which  melts  at 
175°-176°. 

It  seems  to  exert  a  paralyzing  action  upon  the  nerve-centers  and 
29 


450  CHEMISTRY  OF  THE  LEUCOMAINS. 

heart-ganglia,  and  leaves  the  nerves  and  muscles  unaffected.     0.5  mg. 
kills  10  g.  frogs. 

Spermin,  CgH^N,  or  C^Hj^Ng ,  is  the  basic  substance  obtained  by 
Schreiner  (1878)  from  semen,  calf's  heart,  calf's  liver,  bull's  testi- 
cles, from  the  organs  of  leukemics,  and  also  from  the  surface  of 
anatomical  specimens  kept  under  alcohol.  Poehl  has  found  it  in  the 
testes,  ovaries,  prostate,  thyroid  gland,  pancreas  and  spleen.  In 
1888  Kunz  reported  the  presence  of  a  non-poisonous  base,  CgHgN, 
spermin  or  ethyleneimid  in  cholera  cultures.  In  this  case  it  occurs, 
then  as  a  ptomain.  A  confirmation  of  the  identity  of  the  two  bases 
is  necessary.  Previous  to  this,  however,  it  had  been  known  for  a 
long  time  under  the  name  of  "  Charcot-Neumann  or  Leyden  crys- 
tals," which  are  the  phosphate  of  spermin.  These  peculiarly  shaped 
crystals  have  been  found  in  the  sputa  of  a  case  of  emphysema  with 
catarrh,  in  the  bronchial  discharges  in  acute  bronchitis,  as  well  as  in 
sputa  of  chronic  bronchitis,  in  the  blood,  spleen,  etc.,  of  leucocy- 
themics  and  anemics,  and  in  the  normal  marrow  of  human  bones, 
as  well  as  in  human  semen,  also  in  nasal  secretions  and  in  feces. 
Altogether  it  seems  to  have  a  very  wide  distribution,  especially  in 
certain  diseases,  as  in  leucocythemia. 

Spermatic  stains  yield  with  iodin  the  so-called  Florence's  crystals 
which  resemble  those  of  Teichmann.  These  crystals,  however,  are 
not  characteristic  of  sperma  but  may  be  obtained  from  any  lecithin- 
(choUn)-containing  fluid.  They  have  been  obtained  from  liver  and 
brain  extracts,  and  from  elder  blossoms.  According  to  Bocarius,^ 
the  crystals  represent  a  derivative  of  cholin  and  not  an  iodo-spermin 
as  first  supposed. 

It  can  be  prepared  from  fresh  human  semen  in  the  following 
manner :  The  semen  is  washed  out  of  linen  by  a  little  warm  water, 
evaporated  to  dryness,  boiled  with  alcohol,  and  the  insoluble  por- 
tion is  allowed  to  subside  by  standing  some  hours.  The  precipitate 
is  filtered  off,  washed,  and  dried  at  100°.  This  residue,  containing 
the  spermin  phosphate,  is  triturated,  and  then  extracted  with  warm 
ammoniacal  water.  From  this  solution,  on  slow  evaporation,  the 
phosphate  crystallizes  in  its  peculiarly  shaped  crystals. 

The  free  base  is  obtained,  on  decomposing  the  phosphate  with  baryta 
and  evaporating  the  filtrate,  as  a  colorless  liquid  which  on  cooling 
crystallizes.  From  alcohol  it  crystallizes  in  wavellite-shaped  crystals 
which  readily  absorb  water  and  carbonic  acid  from  the  atmosphere. 
They  are  readily  soluble  in  water  and  in  absolute  alcohol,  almost  in- 
soluble in  ether,  and  possess  a  strong  alkaline  reaction.  When 
heated  on  a  platinum  foil  it  gives  off  thick,  white  fumes  and  a  weak 
ammoniacal  odor.  With  potassium  bismuth  iodid  it  yields  orange- 
colored  crystalline  floccules,  which  under  the  microscope  appear  as 
»Zeite.  ]physiol.  Chem.,  34,  339. 


SPERMIN.  451 

long,  sharp,  plumose  needles — distinction  from  diethylenediamin. 
The  aqueous  solution  of  the  base  is  precipitated  by  phosphomolybdic, 
phosphotungstic  and  tannic  acids,  gold  and  platinum  chlorids.  It 
cannot  be  volatilized  from  alkaline  solution  by  steam  without  under- 
going decomposition  (Majert  and  Schmidt).     It  is  not  poisonous. 

The  hydrochlorid,  CgHjN.HCl  (?),  crystallizes  in  six-sided  prisms, 
united  in  tufts,  and  is  extremely  soluble  in  water,  almost  insoluble 
in  absolute  alcohol  and  ether. 

The  aurochlorid,  C2H5N.HCI.AUCI3  (?),  forms  shining,  golden- 
yellow,  irregular  plates,  and  when  freshly  precipitated  it  is  easily 
soluble  in  water,  alcohol,  and  ether,  but  the  dried  salt  is  incom- 
pletely soluble  in  water.  The  aqueous  solution,  treated  with  mag- 
nesia, gives  off  a  sperm-like  odor.  The  platinochlorid  crystallizes 
in  prisms. 

The  phosphate,  (C^H^NX-HjPO,  +  SHp  (?),  forms  prisms  and 
slender  double  pyramids  arranged  in  rosettes.  It  is  difficultly  sol- 
uble in  hot  water,  insoluble  in  alcohol,  easily  soluble  in  dilute  acids, 
alkalis  and  alkali  carbonates.  It  melts  with  decomposition  at  about 
170°.  It  is  probable  that  the  above  formula  does  not  represent  the 
salt  as  found,  and  from  theoretical  considerations  Ladenburg  is 
inclined  to  think  that  Schreiner's  phosphate  has  the  composition 
(C3H,NH),Ca(PO,),. 

Ladenburg  and  Abel  prepared  in  1888  a  compound,  ethyleneimin, 
which  was  first  supposed  to  be  isomeric  with  spermin.  The  reaction 
whereby  it  is  prepared  is  similar  to  that  by  which  Ladenburg 
effected  the  synthesis  of  piperidin.  Ethylenediamin  hydrochlorid  is 
subjected  to  dry  distillation,  when  it  decomposes  into  ammonium 
chlorid  and  the  hydrochlorid  of  the  new  base.  The  reaction  was 
supposed  to  be  represented  by  the  equation  : 


CHaNHj.HCl  CH, 

CH,NH,.HC1     ~      CH. 


\nh.hci  +  nh^ci. 


Since  then  Ladenburg  showed  that  the  boiling  point  of  this 
compound  did  not  agree  with  what  it  should  be  theoretically,  if 
represented  by  the  above  formula.  A  determination  of  the  vapor 
density  showed  that  the  molecular  weight  was  twice  that  correspond- 
ing fo  the  formula  given,  and  hence  was  C^Hj^N^.  Majert  and 
Schmidt  assuming  spermin  to  be  ethyleneimin,  as  was  apparently 
shown  by  Ladenburg  and  Abel's  investigation,  attempted  to  prepare 
the  latter  on  a  manufacturing  scale  with  the  expectation  that  it 
might  be  used  as  a  substitute  for  Brown-Sequard's  testicular  fluid. 
They  were  soon  able  to  show,  however,  that  ethyleneimin  did  not 
possess  the  composition  assigned  to  it,  but  that  it  was  identical  with 
Hofmann's  diethylenediamin  (piperazin,  p.  418), 

HN<  >NH. 


>i 


CHj.CH, 


/' 


462  CHEMISTRY  OF  THE  LEUCOMAINS. 

This  was  soon  confirmed  by  Hofmann  and  by  Ladenburg.  Spermin 
was  then  assumed  to  be  identical  with  piperazin,  but  later  (1891) 
Majert  and  Schmidt  compared  some  spermin  from  Schreiner  with 
their  own  piperazin  and  found  the  two  bases  to  be  distinct,  especially 
with  reference  to  the  phosphate  and  the  potassium  bismuth  iodid 
precipitates.  Poehl  confirmed  the  difference  between  spermin  and 
piperazin. 

On  cleavage  of  casein  with  hydrochloric  acid  Cohn  ^  obtained  a 
substance  which  at  first  he  regarded  as  a  pyridin  derivative,  CgH^NO. 
Later  (1900),  he  gave  up  his  view  as  to  its  pyridin  nature  and 
adopted  the  formula  (CgHjj]SrO)2 ,  that  of  a  leucinimid.  This  body 
as  well  as  the  basic  reduction  product  (CgHj3N)2  he  now  regards  as 
derivatives  of  piperazin  or  diethylenediamin.  The  piperazin  nature 
of  Cohn's  products  is  supported  by  the  work  of  Hoyer.^  Nevertheless 
it  is  true  that  as  yet  there  is  no  evidence  that  piperazin  bases  exist 
preformed  in  the  proteid  molecule.  The  related  pyrimidin  group, 
however,  is  present  (see  p.  418). 

In  1891  Poehl  announced  that  the  composition  of  spermin  was 
more  complex  than  was  formerly  supposed.  He  ascribed  to  it  the 
formula  CioH2gN4 .  The  formula  of  the  platinum  salt  corresponded 
to  CjQH2gN^.4E[C1.2PtCl4;  and  that  of  the  gold  salt  was  represented 
by  CioH2gN,.4HC1.4AuCl3 .     Later  he  gave  the  formula  C^Hj^N^ . 

From  this  it  would  appear  that  spermin  is  essentially  distinct 
from  piperazin.  The  composition  and  structure  of  this  interesting 
base  must  therefore  be  considered  as  not  settled.  Undoubtedly 
bodies  of  entirely  different  composition  have  been  included  under 
the  head  of  spermin.  It  is  quite  probable  that  it  is  related  to 
cadaverin  in  which  case  its  source  might  be  looked  for  in  the  prota- 
min  or  histon  molecule. 

According  to  Poehl,  it  acts  as  a  tonic  on  the  entire  nervous 
system. 

a-Methyl  quinolin,  Cij^H^N ,  is  present  in  part  or  wholly  as  the 
free  base  in  the  secretion  of  the  anal  glands  of  the  skunk  (Mephitis 
mephitica).  It  was  isolated  in  1897  by  Aldrich  and  Jones  ^  and  is 
of  special  interest  since,  with  the  exception  of  the  kynurenic  acid  * 
from  the  dog's  urine,  it  is  the  only  quinolin  compound  isolated 
from  the  animal  body.  The  structure  of  these  two  quinolin  deriva- 
tives is  represented  by  the  formulae  : 

^Zeits.  physiol.  Chem.,  22,  153;  26,  395  ;  29,  283. 
" Zeits.  physiol.  Chem.,  34,  347. 
^Journ.  Exp.  Med.,  2,  439,  1897. 
*Zeits.  physiol.  Chem.,  33,  390,  1901. 


METHYL   QUINOLIN.  463 

H 


HO      C      g.COOH 


N%. 


H  H 

a-METHYL  QUINOLIN.  KYNURENIC  AcID,   OF 

y  OXY  fi  QuiNOLIN  CABBONIC  AcID. 

The  latter  by  splitting  off  the  carboxyl  group  yields  kynurin,  the 
homologue  of  indoxyl.  The  methyl  quinolin  may  be  compared  with 
skatol  or  /9-methyl  indol.  The  proteid  molecule  is  unquestionably 
the  source  of  the  quinolin  as  well  as  of  the  indol  derivatives.  The 
existence  of  pyrrolidin  carbonic  acid  among  the  cleavage  products  of 
proteids  has  been  noted  on  p.  448. 

The  base  is  a  highly  refractive,  colorless  oil  possessing  a  faint 
quinolin  odor  which  is  very  pronounced  on  warming.  It  is  readily 
volatile  with  steam  and  fumes  when  hydrochloric  acid  is  held 
near  it.  It  is  readily  soluble  in  ether,  chloroform,  alcohol  and  in 
mineral  acids  ;  insoluble  in  alkalis  and  sparingly  soluble  in  both  hot 
and  cold  water.  It  forms  addition  products  with  methyl  iodid  and 
with  bromin.     With  phthalic  anhydrid  it  yields  quinaldin  yellow. 

The  platinochlorid,  (CjyHgN.HCl)2PtCl^ ,  forms  characteristic 
yellow  needles  which  melt  at  226°—  230°.  It  is  sparingly  soluble  in 
cold  water  and  on  heating  in  a  test-tube  it  decomposes,  giving 
rise  to  a  volatile  oil  which  on  cooling  yields  transparent  colorless 
needles. 

The  aurochlorid  is  precipitated  as  fine  slender  yellow  needles 
which  melt  at  153°.  It  is  more  soluble  than  the  preceding  com- 
pound.    The  silver  nitrate  compound  forms  colorless  needles. 

The  bichromate,  (C,(,HgN)2.H2CrjOy ,  is  extremely  characteristic 
and  is  precipitated  as  a  yellowish  red  oil  which  promptly  crystallizes, 
forming  needles  which  in  form  and  color  resemble  those  of  the 
platinum  salt.  It  melts  with  decomposition  at  130-140°.  A 
ferrocyanid  can  also  be  prepared. 

The  picrate  forms  yellow  crystals  which  on  rapid  heating  melt  at 
177°. 

The  zinc  chlorid  compound,  (CjoHgN.HCl)2.ZnCl2 ,  according  to 
Aldrich  and  Jones  is  especially  useful  for  the  separation  and  purifi- 
cation of  the  base.  It  is  readily  soluble  in  hot,  insoluble  in  cold 
water.     It  forms  gypsum-like  crystals  which  melt  at  230°- 240.° 

According  to  Cohn  ^  the  injection  of  this  base  into  dogs  gives 
rise  to  an  intense  icterus  which  soon  passes  away.  Rabbits  are  more 
susceptible  and  large  doses  cause  pronounced  hemoglobinuria  and 
eventually  death.  In  neither  animal  does  quinolin  appear  in  the 
urine  showing  that  the  group  is  apparently  destroyed  in  the  body. 

1  Zeiis.  physiol.  Chem.,  20,  210,  1894. 


454  CHEMISTRY  OF  THE  LEUCOMAINS. 

THE  CREATININ  GROUP. 

The  knowledge  of  the  formation  of  basic  substances  (ptomains) 
during  the  putrefaction  of  nitrogenous  organic  matter  led  to  a  series 
of  investigations  having  for  their  object  the  isolation  of  alkaloidal 
bodies,  if  such  existed,  from  the  normal  living  tissues  of  the  organ- 
ism. A  number  of  basic  compounds,  such  as  the  purin  bases  al- 
ready described,  had  been  known  for  a  long  time,  although  their 
physiological  relation  to  the  animal  economy  was  little,  if  at  all, 
imderstood.  Guareschi  and  Mosso,  in  the  course  of  their  researches 
on  ptomains,  were  among  the  first  to  direct  their  attention  to  the 
possible  presence  of  ptomain-like  bodies  in  fresh  tissues.  They  ob- 
tained in  those  cases  where  the  extraction  was  carried  on  without 
the  use  of  acids  only  very  minute  traces  of  an  alkaloidal  body  (pos- 
sibly cholin),  and  an  inert  substance,  methyl  hydantoin,  which, 
although  it  can  scarcely  be  classed  as  a  basic  compound,  is  closely 
related  to  creatin,  and  to  methyl  guanidin  (page  284),  and  for  this 
reason  will  be  described  at  the  end  of  this  section.  Other  Italian 
chemists,  as  Paterno  and  Spica  and  Marino-Zuco,  had  also  shown 
that  the  normal  fluids  and  tissues  of  the  body  were  capable  of  yield- 
ing substances  alkaloidal  in  nature,  and  these  were  regarded  by  them 
as  identical  with,  or  similar  to,  the  ptomains  of  Selmi. 

Gautier  devoted  much  time  to  the  study  of  the  leucomams  occur- 
ring in  fresh  muscle  tissue,  and  he  succeeded  in  isolating  the  several 
compounds  presently  to  be  described. 

A  number  of  these  substances  are  credited  with  possessing  an  in- 
tensely poisonous  action,  and  if  this  is  the  case,  it  is  very  evident 
that  any  undue  accumulation  of  such  bases  in  the  system,  resulting 
either  from  an  interference  in  the  elimination  or  lack  of  destruction, 
may  give  rise  to  serious  disturbances.  The  amount  of  these  substances 
present  in  the  urine  is  said  to  be  very  small — so  small,  indeed,  that  we 
must  rather  look  upon  this  minute  quantity  as  having  escaped  oxi- 
dation in  the  body.  It  is  well  known  that  the  living  tissues  possess 
an  enormous  oxidizing  and  reducing  power,  and,  according  to  Gau- 
tier, there  is  constantly  going  on  in  the  normal  tissues  of  the  body  a 
cycle  of  changes — the  formation  of  leucomains  and  their  subsequent 
destruction  by  oxidation,  before  they  have  accumulated  in  sufficient 
quantity  to  produce  poisonous  effects.  This  formation  and  destruc- 
tion of  bases  in  the  body  has  been  shown  to  occur  in  the  case  of  the 
purin  bodies  (page  343)  and  obviously  intermediate  cleavage  prod- 
ucts in  proteid  and  other  metabolisms  are  being  constantly  formed 
to  be  converted  promptly  into  other  and  more  simple  bodies.  Cre- 
atin and  creatinin  are  examples  of  this  kind,  and  Gautier's  bases 
show  more  or  less  relation  to  these  substances.  It  is  eminently  de- 
sirable, however,  that  the  results  obtained  by  Gautier  and  his  co- 
workers be  confirmed,  if  possible,  in  view  of  the  reasonable  doubt 


THE  CREATININ  GROUP.  465 

that  may  be  raised  as  to  the  chemical  individuality  or  purity  of  some 
of  these  compounds.  The  study  of  the  hexon  bases  and  of  the  mon- 
amino-  and  di-amino  acids  in  general  make  such  a  revision  all  the 
more  necessary. 

The  following  method  was  employed  by  Gautier  in  his  study  of 
the  leucomains  of  muscle  tissue  :  The  finely  divided  fresh  beef-meat 
or  the  Liebig's  meat  extract  is  treated  with  twice  its  weight  of  water, 
containing  0.25  gram  of  oxalic  acid,  and  one  to  two  c.c.  of  com- 
mercial peroxid  of  hydrogen  per  liter.  The  purpose  of  these  pre- 
cautions is  to  prevent  fermentation.  Inasmuch  as  peroxid  of  hy- 
drogen is  not  without  action  on  organic  substances  its  use  should 
be  avoided.  At  the  end  of  twenty-four  hours  the  liquid  is  raised  to 
the  boiling  point,  then  filtered  through  linen,  and  the  residue  is 
thoroughly  squeezed.  The  filtrate  is  again  raised  to  the  boiling 
point  in  order  to  coagulate  any  remaining  albumin,  and  finally 
filtered  through  paper.  The  clear  liquid  thus  obtained  is  evaporated 
in  a  vacuum  at  a  temperature  not  exceeding  50°,  and  the  acid  syrupy 
residue  is  extracted  with  99  per  cent,  alcohol  ;  the  alcoholic  extract 
is  in  turn  evaporated  in  a  vacuum,  and  the  residue  taken  up 
with  warm  alcohol  of  the  same  strength.  The  filtered  alcoholic 
solution  is  set  aside  for  twenty-four  hours,  and  any  deposit  which 
forms  is  removed  by  filtration  ;  ether  (65°)  is  then  added  as  long  as 
a  precipitate  continues  to  form,  and  the  whole  is  again  allowed  to 
stand  for  twenty-four  hours.  The  ether-alcoholic  filtrate  from  this 
precipitate  is  evaporated  first  on  the  water-bath,  and  finally  in  a 
vacuum ;  the  slight  residue  obtained  contains  a  small  quantity  of 
basic  substances  possessing  the  odor  of  hawthorn. 

The  syrupy  precipitate  produced  by  the  ether  partially  crystallizes 
on  standing  ;  a  little  absolute  ether  is  then  added,  and  after  standing 
several  days  more  the  liquid  is  separated  by  means  of  an  aspirator 
from  the  deposit  of  crystals  (A).  These  are  first  washed  with  99 
per  cent,  alcohol,  and  then  extracted  with  boiling  95  per  cent,  alcohol. 
The  alcoholic  solution,  concentrated  by  evaporation,  gives,  on  cool- 
ing, a  deposit  of  lemon  yellow  crystals  of  xantho-creatinin  (B),  from 
the  mother  liquor  of  which  there  separates  a  crop  of  new  crystals 
(C).  The  residue  of  the  crystals  (A)  left  after  treatment  with  the 
boiling  95  per  cent,  alcohol  is  extracted  with  boiling  water,  which 
afterward  gives  a  slight  deposit  of  yellowish  white  crystals  of  amphi- 
creatin  (D).  The  aqueous  mother  liquor  on  concentration  yields 
another  deposit  of  orange  colored  crystals  of  cruso-creatinin  (E). 
Gautier  has,  furthermore,  separated  three  other  bases  from  the 
mother  liquors  of  the  crystals  obtained  as  above.  Thus,  a  base 
which  he  named  pseudoxanthin  is  stated  to  have  been  obtained  by 
evaporating  the  alcoholic  mother  liquors  of  B,  D,  E  (?)  in  a  vacuum, 
taking  up  the  residue  with  water,  and  precipitating  the  hot  solution 
with  copper  acetate.     The  precipitate  is  decomposed  with  hydrogen 


456 


CHEMISTRY  OF  THE  LEUC03IAINS. 


sulphid,  and  the  aqueous  solution,  filtered  while  boiling  hot,  yields  a 
deposit  of  a  sulphur  yellow  powder  of  pseudoxanthin.  Thus,  by  the 
use  of  alcohol,  ether,  and  water,  Gautier,  according  to  his  statement, 
has  succeeded  in  obtaining  a  sharp  separation  between  these  bases. 
The  importance  of  the  subject  is  such  as  to  require  not  only  confir- 
mation of  the  results  arrived  at  by  Gautier,  but  also  a  more  detailed 
and  exact  study  of  the  chemical  and  physiological  behavior  of  these 
bodies. 

The  following  method  was  employed  by  Gautier  and  Landi  in 
1892  in  their  study  of  the  changes  in  meat.  The  meat  extract  was 
concentrated  in  a  vacuum  to  one-eighth  its  volume,  then  cooled, 
precipitated  with  neutral  lead  acetate,  filtered,  and  after  washing  the 
precipitate,  the  filtrate  was  again  concentrated  to  one-half  its  volume, 
and  the  lead  removed  by  hydrogen  sulphid.  The  filtrate  was  again 
concentrated  to  one-half  its  bulk  and  dialyzed.  The  bases  are  pres- 
ent in  the  dialysate.  The  dialyzed  portion  therefore  was  concen- 
trated, acidulated  with  nitric  acid,  and  precipitated  with  phospho- 
molybdic  acid.  The  precipitate  is  collected  and  washed  at  once  with 
very  dilute  nitric  acid,  then  with  water.  It  is  then  boiled  with 
neutral  lead  acetate ;  the  bases  and  the  greater  part  of  the  xanthin 
and  carnin  pass  into  solution.  After  removal  of  the  lead,  the  fil- 
trate is  evaporated  in  a  vacuum,  then  extracted  with  alcohol.  The 
residue  is  examined  for : 

Bases  A. — It  is  treated  with  dilute  ammonia  ;  this  dissolves  xan- 
thin, hypoxanthin,  guanin,  carnin,  etc.,  whereas  creatin,  etc.,  are  in- 
soluble. The  ammonia  is  allowed  to  evaporate,  and  hence  adenin 
and  guanin  separate  out.  Hypoxanthin  and  xanthin  remain  in 
solution. 

Bases  B. — The  alcoholic  filtrate  from  above  residue  is  neutralized, 
concentrated,  and  treated  with  mercuric  chlorid.  The  mercury 
precipitate  is  washed,  decomposed  with  hydrogen  sulphid,  the  solu- 
tion filtered,  and  the  filtrate  is  precipitated  with  copper  acetate  : 

1.  In  the  cold — acids  of  the  carbopyridic  series,  which  are  crys- 
talline and  give  crystalline  platinochlorids. 

2.  In  boiling  solution — xanthin  bases. 

3.  The  portion  not  precipitated  by  cold  or  hot  copper  acetate  is 
the  most  important.  The  copper  is  removed  with  hydrogen  sulphid, 
the  filtrate  evaporated  to  dryness,  and  extracted  with  alcohol — 
guanin,  creatin,  neurin,  chlorin,  butylenediamin,  etc.,  neuridin, 
ethylenediamin  ;  hydropyridin  bases  and  homologues,  and  bases  that 
give  pyrrol  on  distillation  with  lime ;  all  are  very  poisonous. 

Bases  C. — The  mercuric  chlorid  filtrate  is  concentrated  to  drive 
off  the  alcohol  and  the  mercury  removed  with  hydrogen  sulphid. 
Lead  acetate  is  added,  the  liquid  filtered,  and  after  removal  of  the 
lead  is  evaporated  to  dryness  and  extracted  with  dilute  alcohol — 
the    residue  was    creatin ;    the    filtrate   contained  oxy-ethylenamin, 


XANTHO-CREATININ.  457 

methyl  guanidin,  etc.  Almost  all  of  these  are  poisonous.  They 
are  less  abundant  than  the  others. 

(a)  Xanthin  bases.  Exist  in  minute  amount  in  meat  and  are  not 
toxic. 

(6)  Carbopyridic  bases.  Likewise  present  only  in  small  amount. 
They  produce  stupefaction  in  animals,  but  otherwise  are  not 
dangerous. 

(c)  Xeurin  and  hydropyrrolic  bases.  They  are  the  most  abundant 
leucomains  in  meat,  and  are  the  most  poisonous.  Minute  doses  in 
mice  produce  dyspnoea,  spasmodic  movements  of  the  extremities, 
bristling  of  the  hair,  paralysis,  tetanic  convulsions  and  death.  The 
presence  of  neurin  as  a  muscle  constituent  may  well  be  questioned 
especially  since  Gulewitsch  has  been  unable  to  find  it  in  fresh  brains. 

(d)  Creatin  bases.  These  produce  in  mice  vomiting,  diarrhoea, 
tetanic  convulsions  followed  by  paralysis  of  the  extremities. 

Cruso-creatinin,  C^H^X^O,  forms  orange  yellow  crystals  which 
are  slightly  alkaline  in  reaction  and  possess  a  somewhat  bitter  taste. 
It  yields  a  soluble,  non-deliquescent  hydrochlorid  crystallizing  in 
bundles  of  needles ;  also  a  soluble  platinochlorid  which  forms  tufts 
of  beautiful,  slender  prisms.  The  aurochlorid  is  obtained  as  slightly 
soluble,  crystalline  grains,  and,  like  the  platinum  double  salt,  is 
partially  decomposed  on  heating.  It  is  not  precipitated  by  acetate 
of  zinc  or  by  mercuric  nitrate,  but  is  precipitated  in  the  cold  by 
solutions  of  alum.  Zinc  chlorid  produces  in  somewhat  concentrated 
solutions  a  pulverulent  precipitate  which  dissolves  on  heating,  and 
recrystallizes  again  on  cooling.  Like  xantho-creatinin,  it  is  not 
thrown  out  of  solution  by  oxalic  or  nitric  acid,  and  is  thus  dis- 
tinguished from  urea  and  guanidin ;  nor  is  it  precipitated  by  acetate 
of  copper — a  distinction  from  xanthin  derivatives.  Mercuric  chlorid 
produces  an  abundant  flocculent  precipitate  which  on  heating  partially 
dissolves,  decomposing  at  the  same  time.  Sodium  phosphomolybdate 
gives  a  heavy  yellow  precipitate,  whereas  potassium  mercuric  chlorid 
and  iodiu  in  potassium  iodid  have  no  effect.  Potassium  ferricyanid 
is  not  reduced.  This  base  differs  in  its  composition  from  creatinin 
by  HCX,  the  elements  of  hydrocyanic  acid,  but  in  its  crystalline 
form  and  alkaline  reaction,  and  some  other  properties,  it  would  seem 
to  be  closely  related  to  this  latter  substance.  Because  of  this  ap- 
parent relationship  and  its  golden-yellow  color,  Gautier  named  it 
cruso-creatinin. 

Xantho-creatimii,  C^Hj^X^O,  is  said  to  be  the  most  abundant  of 
muscle  leucomains.  It  crystallizes  in  sulphur  yellow,  thin  spangles, 
consisting  of  nearly  rectangular  plates  which  resemble  somewhat  those 
of  cholesterin.  It  is  soft  and  talc-like  to  the  touch  ;  possesses  a 
slightly  bitter  taste,  and  when  dissolved  in  boiling  alcohol  it  gives 


458  CHEMISTRY  OF  THE  LEUCOMAINS. 

off  the  odor  of  acetamid,  though  ordinarily  in  the  cold  it  has  a  slight 
cadaveric  odor.  When  heated,  the  substance  evolves  an  odor  of 
roast  meat,  carbonizes  in  part,  and  yields  ammonia  and  methylamin. 
The  crystals  are  amphoteric  in  reaction,  are  soluble  in  cold  water, 
and  can  be  recrystallized  from  boiling  99  per  cent,  alcohol. 

It  forms  a  hydrochlorid  crystallizing  in  plumose  needles,  and  a 
very  soluble  platinochlorid ;  the  aurochlorid  crystallizes  with  diffi- 
culty. Like  creatinin,  it  is  precipitated  by  zinc  chlorid ;  the  yel- 
lowish white  precipitate  dissolves  with  partial  dissociation  on  warm- 
ing, and  on  cooling  separates  as  isolated  or  stellate  groups  of  fine 
needles  which  possess  the  composition  (C5HjQN^O)2ZnCl2 .  Silver 
nitrate  throws  down,  in  the  cold,  a  flocculent  precipitate  which  like- 
wise dissolves  on  heating,  and  recrystallizes  in  needles.  Mercuric 
chlorid  produces  a  yellowish  white  precipitate.  It  is  not  precipitated 
by  oxalic  or  nitric  acid,  nor  by  potassium  mercuric  chlorid,  or  iodin 
in  potassium  iodid.  Tannin  produces  in  time  a  slight  turbidity, 
while  sodium  phosphomolybdate  gives  a  heavy  yellowish  precipitate. 
This  base  is  distinguished  from  the  members  of  the  acid  group  by 
not  giving  a  precipitate  with  copper  acetate,  even  on  heating. 

On  gentle  oxidation  with  potassium  permanganate  it  is  converted 
into  a  black  substance  insoluble  in  acids  and  alkalis,  and  resembling 
azulmic  acid.  By  treatment  with  recently  precipitated  mercuric 
oxid  it  yields  a  substance  which  can  be  recrystallized  from  boiling 
93  per  cent,  alcohol  in  needles  which  possess  a  slight  alkaline  reac- 
tion, and  forms  a  slightly  soluble,  crystalline  platinochlorid.  This 
new  substance  is  precipitated  from  alcoholic  solution,  by  the  addi- 
tion of  ether,  as  a  mass  of  beautiful,  white  silky  needles  resembling 
caffein.     These  crystals  melt  at  174°  ;  caffein  melts  at  178°. 

Xantho-creatinin,  given  in  fairly  large  doses,  is  poisonous,  pro- 
ducing in  animals  depression,  somnolence,  and  extreme  fatigue, 
accompanied  by  frequent  defecation  and  vomiting.  In  its  general 
properties  this  base  resembles  creatin  very  much,  and  it  was  on 
account  of  this  resemblance  and  its  yellow  color  that  it  was  named 
xantho-creatinin.  This  relation  becomes  especially  evident  since 
the  base  appears  in  the  physiologically  active  muscle  at  the  same 
time  with  creatinin,  sometimes  in  about  one-tenth  of  the  quantity  of 
the  latter.  Monari  found  this  base  in  the  aqueous  extract  of  the 
muscles  of  an  exhausted  dog,  and  also  in  the  urine  of  soldiers  tired 
by  several  hours'  march.  He  also  claimed  to  have  demonstrated  its 
presence  in  the  urine  of  a  dog  after  previous  injection  of  creatinin. 
Stadthageu  was  not  able  to  isolate  this  base  from  his  urine  after  pro- 
longed muscular  exercise,  and  arrived  at  the  conclusion  that  it  does 
not  occur  in  urine,  and  that  Monari's  base  was  an  impure  crea- 
tinin. Colasanti,  in  1884,  and  again  in  1891,  isolated  from  lion's 
urine  by  Neubauer's  zinc  chlorid  method  for  creatinin  the  latter 
compound  and   a  yellow  body  which  crystallized  as  canary-yellow, 


AMPHI-CREATIN.  459 

small,  opaque  scales,  or  as  small,  orange  yellow,  granular  masses 
composed  of  needle-shaped  crystals.  This  he  considered  to  be  xantho- 
creatinin,  derived  from  the  large  excess  of  creatinin  in  the  food. 

Amphi-creatin,  CgHjgN^O^,  is  slightly  soluble  and  crystallizes 
from  boiling  water  into  yellowish  white  oblique  prisms,  which 
possess,  if  any,  a  slightly  bitter  taste.  When  heated  to  100°  it 
decrepitates  somewhat,  and  at  110°  it  becomes  opaque  white. 
Potassium  hydrate  does  not  decompose  it  in  the  cold.  Although 
a  weak  base,  it  combines  to  form  salts  just  as  the  preceding  mem- 
bers of  this  group.  The  hydrochlorid  is  crystalline,  and  is  not 
deliquescent;  the  platinochlorid  forms  rhombic  plates,  which  are 
soluble  in  water,  but  are  insoluble  in  absolute  alcohol ;  the  auro- 
chlorid  crystallizes  in  easily  soluble,  very  small,  microscopic  crystals, 
which  are  tetrahedral  to  hexahedral  in  their  habit.  It  is  not  pre- 
cipitated by  copper  acetate  or  by  mercuric  chlorid ;  nor  does  it  give 
the  murexid  test,  or  the  xanthin  reaction.  Sodium  phosphomolyb- 
date  produces  a  yellow,  pulverulent  precipitate.  In  its  properties  it 
resembles  creatin,  and  indeed  Gautier  thinks  it  may  be  possibly  a 
combination  of  creatin,  C^HgNgOg,  and  a  base,  CgH^^N^Og,  which,  it 
will  be  seen,  differs  from  the  former  only  by  a  HON  group.  This 
second  compound,  if  it  really  exists,  has  an  analogy  in  cruso-creatinin, 
the  relation  of  which  to  creatinin  may  be  expressed  by  the  equation : 

C^HgNp  =  C,H^N30  +  HCN. 

Crubo-crkatinin.       Creatinin. 

In  a  similar  manner,  amphi-creatin  may  be  regarded  as 
CgH,,N,0,=  2C,H,N30,4-HCN. 

Amphi-creatin.  Creatin. 

A  Base,  CuHj^Nj^Og,  was  isolated  by  Gautier  from  the  mother 
liquors  of  xantho-creatinin.  It  crystallizes  in  colorless  or  yellowish, 
thin,  apparently  rectangular  plates,  which  are  tasteless  and  possess 
an  amphoteric  reaction.  The  hydrochlorid  forms  bundles  of  fine 
needles  ;  the  sulphate  yields  a  confused  mass  of  needles  ;  the  platino- 
chlorid is  soluble,  non-deliquescent  and  crystalline.  When  heated 
with  water  in  a  sealed  tube  at  180°- 200°  it  gives  off  ammonia  and 
carbonic  acid,  and  is  converted  into  a  new  base,  which,  however,  has 
not  been  studied.     This  reaction  may  be  expressed  by  the  equation  : 

C,,H,,N,,0,  =  2C,H,„N  A  +  CO(NH,),. 

Urea. 

The  urea  which  at  first  forms  is,  in  turn,  decomposed  thus  : 
CO(NH,),  +  HP  =  CO,  -h  2NH3. 


460  CHEMISTRY  OF  THE  LEUGOMAINS. 

It  is  to  be  observed  that  this  base  differs  in  composition  from  the 
following  one  by  HON,  the  hydrocyanic  acid  molecule. 

A  Base,  Cj2H25Nj^05,  was  obtained  from  the  mother  liquors  of 
cruso-creatinin,  and  forms  rectangular  silky  plates,  resembling  those 
of  the  preceding  base  and  of  xantho-creatinin.  It  forms  crystalliz- 
able  salts. 

These  complex  bases,  as  already  pointed  out,  require  further  study 
in  order  to  elucidate  their  chemical  nature.  The  equations  given 
above,  while  they  harmonize  with  Gautier's  view  as  to  the  r6le  of 
HON  cannot  be  looked  upon  as  established  facts.  On  the  contrary 
they  are  suggestive  of  the  impurity  of  the  substances  examined. 

/N(CH3).CHj 

Methyl  hydantoin,  c,H.N A   =   ^K^h— co 

This  substance  was  obtained  by  Guareschi  and  Mosso  (1883),  by 
extracting  fresh  meat  with  1-1.5  volumes  of  water  (without  addition 
of  acid),  for  two  hours  at  50°  -  60°.  The  aqueous  extract  was  evap- 
orated on  a  water-bath  and  the  residue  was  extracted  with  95  per 
cent,  alcohol.  This  alcoholic  solution,  after  the  alcohol  was  driven 
off,  was  first  taken  up  in  water,  filtered,  and  the  aqueous  solution 
was  extracted  with  ether,  then  rendered  alkaline  with  ammonia, 
and  again  extracted  with  ether.  The  alkaline  ether  extract  gave  on 
evaporation  a  white  crystalline  residue  of  methyl  hydantoin.  The 
amount  of  this  substance  present  in  flesh  appears  to  be  quite  vari- 
able, since,  at  times,  none  whatever  can  be  extracted.  Albertoni 
isolated  it  from  a  dog's  flesh.  Previous  to  its  discovery  in  flesh  by 
Guareschi  and  Mosso,  it  was  known  for  a  long  time  as  a  decompo- 
sition product  of  various  nitrogenous  bases  of  the  body.  Thus, 
Neubauer  prepared  it  by  heating  creatinin  with  barium  hydrate, 
while  Huppert  obtained  it  by  fusing  together  sarcosin  with  urea.  As 
it  occurs  in  muscle,  it  is  probably  derived  from  the  creatin,  though 
under  what  conditions  this  splitting  up  takes  place  is  not  definitely 
known.  It  may  be  due  to  an  enzyme  or  to  nitrous  acid  in  which 
case  the  change  would  correspond  to  the  conversion  of  guanin  into 
xanthin.  Acetic  and  lactic  acids  are  incapable  of  effecting  this  change. 
At  all  events,  it  belongs  to  the  ureids,  and  is  intermediate  between 
creatinin,  sarcosin,  and  urea.  Compare  the  above  formula  with  that 
of  creatinin  and  methyl  guanidin  (p.  284). 

Methyl  hydantoin  forms  prisms  which  are  easily  soluble  in  water 
and  alcohol,  and  but  slightly  soluble  in  cold  ether.  It  melts  at 
156°  (Salkowski);  at  159°- 160°  (Guareschi  and  Mosso).  Its 
aqueous  solution  is  slightly  acid  in  reaction.  On  strong  heating  it 
volatilizes.  When  fused  with  potassium  hydrate  it  gives  off  am- 
monia :    it  reduces   mercuric  nitrate  in   the  cold.      Treated   with 


LEUCOMA'iNS  OF  EXPIRED  AIR.  461 

mercuric  oxid  it  assumes  an  alkaline  reaction,  and  the  filtrate  on 
heating  yields  metallic  mercury.  With  silver  oxid  it  forms  pearly 
lanceolate  plates  having  the  composition  C^HgNgOj.Ag.  It  does 
not  give  the  alkaloidal  reactions. 

UNDETERMINED  LEUCOMAINS. 

Leucomains  of  Expired  Air. 

It  was  shown  at  quite  an  early  period  that  exhalations  from  ani- 
mals contain,  besides  an  increased  amount  of  carbonic  acid,  some 
organic  matter,  the  nature  of  which,  on  account  of  the  exceedingly 
minute  quantity  in  which  it  occurs,  has  never  been  satisfactorily 
determined.  Ransome,  in  1870,  estimated  the  organic  matter  in 
expired  air  by  permanganate  of  potash  to  be  about  0.2  g.  per  day. 
Later  Uffelmann  showed  that  the  amount  of  the  organic  matter  in 
occupied  closed  rooms  increased  in  almost  the  same  ratio  as  carbonic 
acid.  Herrmanns,  however,  denied  the  existence  of  organic  sub- 
stances in  the  expired  air.  Nevertheless,  various  observers  did  not 
hesitate  to  ascribe  to  it  the  ill  effects  consequent  upon  breathing  im- 
pure air,  while  at  the  same  time  the  carbonic  acid  formed  during 
respiration  was  considered  as  either  entirely  inert  or  as  insignificant 
in  its  action.  Thus,  respired  air  from  which  moisture  and  carbonic 
acid  have  been  removed,  but  which  still  contains  the  organic  vapors, 
was  found  by  some  to  be  highly  poisonous.  On  the  other  hand,  if  the 
respired  air  is  drawn  through  a  red-hot  tube,  to  destroy  the  organic 
matter,  the  air  thus  purified  is  capable  of  sustaining  life  even  in 
presence  of  a  large  percentage  of  carbonic  acid.  While  it  cannot 
be,  therefore,  doubted  that  the  organic  matter  of  expired  air  plays  a 
most  important  part  in  producing  the  well  known  noxious  effects 
resulting  from  breathing  confined  and  vitiated  air,  nevertheless  it 
would  seem  from  experiments  made  by  Angus  Smith  that  the  increase 
of  even  such  small  quantities  of  carbonic  acid  in  the  air  as  from  0.04, 
the  normal  amount  present,  to  0.1  per  cent.,  is  capable  of  producing 
systemic  disturbances  characterized  by  a  decrease  in  the  pulse  rate 
and  an  increase  in  the  rate  of  respiration. 

Smith  was  consequently  of  the  opinion  that  the  constant  lowering 
of  the  pulse  in  impure  air,  occasioned  by  the  presence  of  carbonic 
acid,  must  have  a  depressing  effect  on  the  vitality.  Whatever  ill 
effects  the  carbonic  acid  may  produce  of  itself,  it  remained  quite  cer- 
tain that  this  gas  was  not  the  most  potent  and  most  injurious  con- 
stituent of  respired  air  ;  and  the  investigations  of  Hammond,  Nowak, 
Seegen,  and  others  pointed  to  the  organic  matter  as  the  direct  and 
immediate  agent  which  produces  those  symptoms  of  sickness  and 
nausea  experienced  in  badly  ventilated  closed  rooms. 

Of  special  importance  to  the  sanitarian  and  physician  is  the  work 
on  the  nature  and  action  of  the  poisonous  principle  of  expired  air 


462  CHEMISTRY  OF  THE  LEUGOMAINS. 

which  was  done  bv  Brown-S6quard,  d'Arsonval,  and  R,  Wurtz. 
The  first  two  observers  found  that  the  vapors  exhaled  by  dogs,  when 
condensed,  and  the  aqueous  liquid  (20-44  c,c.)  thus  obtained  was 
injected  into  other  animals,  death  was  produced,  generally  within 
twenty-four  hours.  The  symptoms  observed  were  dilatation  of  the 
pupil,  increase  of  heart-beat  to  240-280  per  minute,  which  may  last 
for  several  days  or  even  weeks,  while  the  temperature  remains 
normal ;  the  respiratory  movements  were  generally  slowed,  and 
usually  paralysis  of  the  posterior  members  was  observed.  Choleraic 
diarrhoea  was  invariably  present.  As  a  rule,  larger  doses  caused 
labored  respiration,  violent  retching,  and  contraction  of  the  pupil. 
A  rapid  lowering  of  temperature,  0.5°  to  5°,  was  sometimes 
observed.  These  same  symptoms,  apparently  in  aggravated  form, 
were  obtained  when  the  liquid  had  been  previously  boiled  for  the 
purpose  of  destroying  any  germs  that  might  be  present.  The  appear- 
ances presented  on  post-mortem  were  much  like  those  observable  in 
cardiac  syncope. 

The  above  work  was  confirmed  in  part  by  R.  Wurtz,  who,  by 
passing  expired  air  through  a  solution  of  oxalic  acid,  obtained, 
besides  ammonia,  a  volatile  organic  base  which  was  precipitated  by 
Bouchardat's  reagent  and  by  potassio-mercuric  iodid.  It  is  said  to 
form  a  platinum  double  salt  crystallizing  in  short  needles,  and  a 
soluble  gold  salt.  When  heated  to  100°  it  gives  off"  a  peculiar  odor. 
This  basic  substance  may  properly  be  looked  upon  as  a  leucomain. 
The  possibility  of  its  being  an  ammonium  compound  is  not  excluded. 

Dastre  and  Loye,  Lehmann  and  Jessen,  Geyer,  and  Merkel  have 
repeated  the  above  experiments  with  wholly  negative  results. 
Similar  negative  results  were  obtained  by  Hoffmann- Wellenhof  and 
by  Russo-Griliberti  and  Alesi,  who  injected  the  condensed  moisture 
from  expired  air  without  effect.  Ben,  in  1893,  studied  the  subject 
of  the  toxicity  of  expired  air.  From  about  3000  liters  of  his  expired 
air  (eight  hours)  he  obtained  about  100  c.c.  of  condensed  water  hav- 
ing a  peculiar,  not  unpleasant  odor.  It  gave  a  distinct  reaction  for 
ammonia  with  Nessler's  reagent,  but  contained  no  alkaloids.  The 
organic  substance  amounted  to  5  mg.,  or  for  twenty-four  hours  to 
15  mg.  By  repeating  Wurtz's  experiment  with  500  and  700  liters 
of  expired  air  no  alkaloidal  reactions  were  obtained  nor  were  any 
effects  produced  in  animals.  From  these  and  other  experiments,  he 
concluded  that  the  organic  matter  of  expired  air  cannot  induce  acute 
intoxication.  The  dyspnoea  observed  in  confined  spaces  is  due  to 
the  lack  of  oxygen.  Carbonic  acid  may  give  rise  to  dullness  and 
headache,  but  the  amount  may  rise  considerably  and  be  harmless  so 
long  as  oxygen  is  not  decreased  too  much. 

Billings,  Mitchell  and  Bergey  on  examining  the  water  of  conden- 
sation from  expired  air  obtained  traces  of  ammonia  but  no  reactions 
for  alkaloids.     The  fatal  results  met  with  in  Brown-S4quard's  ex- 


LEUCOMAi'NS  OF  THE   URINE.  463 

periments  they  ascribe  to  lack  of  oxygen  and  to  increase  of  carbonic 
acid.  On  the  other  hand  Formanek  explains  the  presence  of 
ammonia  as  due  to  external  decompositions  and  the  fatal  results  as 
due  to  the  combined  effects  of  ammonia  and  carbonic  acid. 

The  most  recent  contribution  on  this  subject  is  that  of  Sanarelli 
and  Biffi.^  These  investigators  demonstrate  that  many  intestinal 
products  may  be  absorbed  and  subsequently  be  eliminated  by  the 
lungs.  When  rectal  injections  of  ammonia,  hydrogen  sulphid, 
butyric  acid,  aceton,  and  carbonic  acid  were  made  these  substances 
could  be  detected  in  the  exhaled  air. 

Sewer  air,  according  to  observations  made  by  Odling,  contains  a 
basic  substance  which  is  probably  a  compound  ammonia.  It  contains, 
however,  more  carbon  than  methylamin  and  less  than  ethylamin. 

It  should  be  remarked  that  Jackson  (1887)  announced  the  pres- 
ence in  expired  air  of  quantities  of  carbon  monoxid  gas  sufficient  to 
produce  the  ill  effects  ordinarily  attributed  to  the  organic  matter. 
The  presence  of  this  poisonous  gas  must  first  be  fully  demonstrated 
before  it  can  be  taken  into  account  in  the  consideration  of  the  tox- 
icity of  air ;  certainly,  even  if  present,  it  cannot  explain  the  results 
obtained  by  the  French  investigators  as  stated  above. 

According  to  Ilosva,  expired  air  contains  nitrous  acid.  This  may 
possibly  be  derived  from  that  which  is  constantly  being  formed  in 
the  mouth,  either  by  the  reduction  of  nitrates  (Miller)  or  by  surface 
action. 

Leucomains  of  the  Urine. 

A  number  of  basic  substances  have  been  isolated  at  different  times 
from  the  urine,  and  on  that  account  they  may  be  properly  classed  as 
leucomains.  Thus,  Liebreich  (1869)  found  in  the  urine  a  base  which 
apparently  was  an  oxidation  product  of  cholin,  and  which  has  since 
been  regarded  as  identical  with  betain.  In  1866  Dupr6  and  Bence 
Jones  found,  among  other  things  in  the  urine,  an  alkaloidal  body 
which  in  sulphuric  acid  solution  possessed  a  blue  fluorescence  (see 
p.  41).  Most  of  the  members  of  the  purin  group  have  been  de- 
tected in  the  urine  and  on  account  of  their  well  defined  nature  they 
are  described  by  themselves.  It  is  desirable  perhaps  in  this  connec- 
tion to  emphasize  the  fact  already  brought  out  (p.  405)  that  the 
greater  part  of  the  purin  bases  found  in  the  urine  are  derived  from 
the  preformed  bases  in  the  food.  Rachford's  view  that  migraine  is 
due  to  intoxication  with  paraxanthin  and  allied  bodies  has  been 
referred  to  on  page  403. 

In  1879,  Thudichum  announced  the  presence  in  the  urine  of  four 
new  alkaloids,  one  of  Avhich,  urotheobromin,  was  subsequently  redis- 
covered by  Salomon  andnamed  paraxanthin.  Another  base  which  was 
obtained,  namely,  reducin,  yielded   a  barium  salt  which  readily  re- 

^Aimali  d'Igiene  sper.,  12,  90,  1902. 


464  CHEMISTRY  OF  THE  LEVGOMAINS. 

duced  the  salts  of  silver  and  mercury.  Its  formula  probably  corres- 
ponded to  CjgHg^NgOg  or  CgH^^NgO^.  A  third  alkaloid,  parareducin, 
formed  a  zinc  compound  having  the  composition  CgHgNgO.ZnO. 
A  fourth  base  is  said  to  give  a  compound  with  platinum  chlorid  and 
to  contain  an  aromatic  nucleus  (aromin).  Besides  these  four  bases 
Thudichum  described  two  other  substances  which  he  considered  to 
be  basic.  These  were  urochrome,  the  normal  pigment  of  the  urine, 
and  creatinin. 

In  1880,  Pouchet  announced  the  presence  of  carnin,  CyHgN^Oj. 
Kriiger  and  Salomon  did  not  meet  with  carnin  in  their  exhaustive 
study  of  the  purin  bases  in  urine  and,  moreover,  they  question  the 
correctness  of  Pouchet's  finding.  The  latter  also  reported  another 
base  which  he  subsequently  analyzed  and  found  to  have  either  the 
composition  C^HjgN^Og  or  C^IIj^N^Og.  This  substance  formed  de- 
liquescent fusiform  crystals,  sometimes  grouped  in  bundles  or 
irregular  spheres.  It  possessed  a  slight  alkaline  reaction  and 
combined  with  acids  to  form  crystallizable  salts.  It  was  soluble  in 
dilute  alcohol,  almost  insoluble  in  strong  alcohol,  insoluble  in  ether. 
The  hydrochlorid  yielded  double  salts  with  gold  chlorid,  platinum 
chlorid,  and  mercuric  chlorid.  The  platinochlorid  formed  deliques- 
cent golden-yellow  rhombic  prisms.  This  base  occurred  in  the 
dialysate  (see  page  320).  From  the  non-dialyzable  portion,  Pouchet 
obtained  another  base  corresponding  to  the  formula  CglljNOj,  which 
he  called  the  "  extractive  matter  of  urine."  It  gave  precipitates 
with  the  general  alkaloidal  reagents,  was  non-cry stallizable,  altered 
on  exposure  to  air  and  was  resin ified  by  hydrochloric  acid.  On 
the  addition  of  platinum  chlorid  it  rapidly  oxidized,  but  did  not 
yield  a  platinochlorid.  The  bases  were  poisonous  to  frogs  ;  produced 
paralysis,  loss  of  reflexes,  and  stoppage  of  the  heart  in  systole.  The 
same  author  regarded  the  urine  as  containing  very  small  quantities 
of  some  pyridin  bases,  analogous  to  or  identical  with  those  obtained 
by  Gautier  and  Etard  from  decomposing  fish. 

Baumstark  isolated  a  compound  from  the  urine  having  the  compo- 
sition C3HgN20.  In  forty  liters  of  urine  it  could  just  be  detected  ; 
was  more  abundant  in  one  case  of  icterus.  It  was  not  present  in 
dog's  urine,  except  after  feeding  benzoic  acid.  It  crystallized 
from  water  in  white  prisms  resembling  hippuric  acid.  The  crystals 
decrepitate  on  heating ;  are  unchanged  at  250°,  but  at  higher  temper- 
ature give  off  dense,  white  vapors  having  a  peculiar  odor  ;  melt  and 
take  fire.  The  odor  is  that  of  burned  horn.  It  is  rather  easily 
soluble  in  hot  water,  difficultly  in  cold  water  and  in  alcohol ;  not  in 
absolute  alcohol  or  ether.  The  solutions  are  neutral.  It  forms 
easily  soluble  salts.  The  hydrochlorid,  CgllgNjO.HCl,  crystallizes 
difficultly  in  dendritic  masses ;  is  deliquescent  and  soluble  in  alcohol. 
It  does  not  combine  with  bases,  and  is  precipitated  by  mercuric  nitrate, 
thus  resembling  allantoin  and  urea.     When  heated  in  a  glass  tube 


LEUCOMAINS  OF  THE   URINE.  465 

or  with  soda-lime,  it  gives  off  a  combustible  gas  having  the  odor  of 
ethylamin.  On  boiling  with  baryta  or  ammonia,  ethylamin  and  bar- 
ium carbonate  result.  With  nitrous  acid  it  gives  sarcolactic  acid. 
A  somewhat  similar  substance  was  isolated  by  Meissner  from  the 
urine  of  the  dog. 

The  distinguished  Italian  toxicologist  Selmi  was,  perhaps,  the  first 
to  draw  attention  to  the  probable  formation  of  basic  substances  in 
the  living  body  during  those  pathological  changes  brought  on  by 
the  presence  of  pathogenic  germs.  In  a  memoir  presented  to  the 
Academy  of  Sciences  of  Bologna,  in  December,  1880,  he  announced 
that  infectious  diseases,  or  those  in  which  there  occurs  an  internal 
disarrangement  of  some  element,  either  plasmic  or  histological,  must 
be  accompanied  or  followed  by  an  elimination  of  more  or  less  char- 
acteristic products  which  would  be  a  sign  of  the  pathological  condi- 
tion of  the  patient.  To  support  this  theory  he  examined  a  number 
of  pathological  urines,  and  succeeded  in  obtaining  from  them  basic 
substances,  some  of  which  were  poisonous,  otherfe  not.  Thus,  a 
specimen  of  urine  from  a  patient  with  progressive  paralysis  gave  two 
bases  strongly  resembling  nicotiu  and  coniin  ;  from  other  patholog- 
ical urines  the  bases  obtained  usually  had  either  an  ammoniacal  or 
trimethylamin  odor.  It  is  well  to  note  that  in  normal  urine  am- 
monia and  trimethylamin  are  present,  while  organic  bases,  as  pepto- 
toxin,  are  absent  (Stadhagen).  Selmi  proposed  to  designate  the  basic 
substances  found  in  disease  as  pathoamins.  The  term  urotoxin  is 
likewise  sometimes  employed  to  designate  the  urine  poison.  A 
strong  confirmation  of  Selmi's  theory  is  seen  in  the  observations 
made  by  Bouchard,  Villi^rs,  Lepine,  Gautier,  and  others,  all  of  whom 
apparently  have  found  basic  substances  in  the  urine  of  various  dis- 
eases. 

Thus,  Bouchard  asserted  the  presence  in  normal  urine  of  two  bases, 
one  soluble  in  ether,  the  other  insoluble  in  ether,  but  soluble  in 
chloroform.  By  the  extraction  of  urine  from  typhoid  fever,  pneu- 
monia, pleuritis,  and  icterus  with  ether  he  obtained  substances  that 
gave  alkaloidal  reactions.  Lepine  and  Guerin  likewise  extracted 
alkaline  urine  with  ether  and  obtained  a  poisonous  substance.  The 
extracts  from  pathological  urines  were  more  poisonous  than  those 
from  normal  urine,  and  the  typhoid  extract  reacted  differently  from 
that  of  pneumonia.  Villi^rs  found  the  basic  substances,  as  a  rule, 
in  pneumonia,  tuberculosis,  abscesses,  but  absent  in  tetanus.  In  all 
these  cases  only  extracts  were  employed,  the  substance  not  being 
isolated  in  a  degree  of  purity  and  in  amount  sufficient  for  analysis. 
It  is  comparatively  easy  (from  the  result  of  the  application  of  the 
so-called  alkaloidal  tests)  to  report  upon  the  presence  of  alkaloids  in 
so  complex  a  fluid  as  the  urine.  It  is  vastly  more  difficult,  however, 
to  isolate  such  bodies  in  a  chemically  pure  condition,  thus  satisfying 
the  requirements  of  exact  research. 
30 


466  CHEMISTRY  OF  THE  LEUCOMAINS. 

The  criticisms  on  these  older  examinations  apply  with  equal  force 
to  many  of  the  more  recent  investigations  of  the  urine  in  disease. 
Thus,  Chiaruttini  applied  Spica's  method  for  the  extraction  of  pto- 
mams  to  the  urine  of  various  nervous  diseases  with  convulsions.  In 
twelve  cases  alkaloids  were  obtained  which  produced  similar  toxic 
effects  in  animals.  Arslan  from  the  urine  of  two  children  with  anky- 
lostomiasis separated  a  toxin  that  induced  anaemia  in  rabbits.  Boinet 
and  Silberet  isolated  three  bases  from  the  urine  of  Basedow's  disease 
that  in  animals  produced  effects  similar  to  those  observed  in  the 
disease.  Marino-Zuco  believed  that  the  poisonous  action  of  extract 
of  the  adrenals,  as  observed  by  Foa  and  Pellacani,  was  due  to  cholin 
(neurin  of  Marino-Zuco).  With  Dutto,  he  found  in  the  urine  of 
Addison's  disease  a  base  which  behaved  with  reagents  like  cholin. 
They  therefore  considered  the  disease  as  a  slow  auto-intoxication  with 
this  base.  It  may  be  mentioned  in  this  connection  that  eclampsia  is 
considered  by  Favre  as  a  ptoma'insemia,  whereas  Bouchard  regards  it 
as  due  to  the  non-elimination  of  the  normal  poisons  of  the  urine. 
There  is  more  reason,  however,  in  considering  it  as  due  to  perverted 
cell  metabolism,  just  as  the  varnishing  of  a  part  or  the  whole  of  the 
skin  results,  as  Kijanitzin  has  pointed  out,  in  the  alteration  of  the 
chemical  products  of  the  underlying  cells.  The  same  author  showed 
that  in  extensive  skin-burns  the  urine,  as  well  as  the  blood  and 
organs,  contains  a  basic  poisonous  substance,  presumably  peptotoxin 
(see  page  328). 

While  there  may  be  doubt  as  to  the  formation  of  a  basic  poison  in 
profound  skin-burns,  there  can  be  no  doubt  from  the  pathological 
changes  as  observed  by  Bardeen  and  by  McCrae  ^  that  toxins,  not 
unlike  those  of  bacterial  origin,  are  produced.  The  destruction  of 
hemoglobin  may  lead  to  the  production  of  histon  which  as  already 
pointed  out  possesses  marked  poisonous  properties. 

In  1889  Luff  examined  the  urine  of  infectious  diseases  for  basic 
products  by  the  following  method  :  A  large  quantity  of  the  urine 
was  rendered  alkaline  with  sodium  carbonate,  and  agitated  vdth  one- 
half  its  volume  of  ether.  After  standing  for  some  time  the  ether 
was  removed,  filtered,  and  then  agitated  with  a  solution  of  tartaric 
acid,  to  remove  the  alkaloids  as  soluble  tartrates.  The  aqueous  acid 
solution  was  then  rendered  alkaline  with  sodium  carbonate,  and  agi- 
tated with  one-half  its  volume  of  ether.  The  ether  was  removed, 
allowed  to  evaporate  spontaneously,  and  the  residue,  after  drying 
over  sulphuric  acid,  was  examined  for  alkaloids. 

The  urine  of  typhoid  fever,  collected  during  a  high  fever  for  four 
days,  gave  a  small  quantity  of  a  white  crystalline  substance.  When 
dissolved  in  hydrochloric  acid  it  gave  reactions  with  phosphomolyb- 
dic  acid,  potassium  mercuric  iodid,  iodin  solution,  tannic  and  picric 
acids,  and  gold  chlorid ;  failed  to  react  with  phosphotungstic  acid 

'  Trans.  Assoc.  Am.  Phys.,  16,  153. 


LEUCOMA'iNS  OF  THE   URINE.  467 

and    platinum    chlorid.     The    examination    of  a  second    case    was 
negative. 

Scarlet  fever  urine  collected  during  the  height  of  the  fever  (four 
gallons)  gave  a  small  amount  of  a  white  semi-crystalline  alkaloid. 
The  solution  in  water  was  faintly  alkaline.  The  hydrochloric  acid 
solution  did  not  react  with  tannic  acid  or  platinum  chlorid,  but  gave 
precipitates  with  the  other  reagents  mentioned  above.  The  amount 
of  substance  was  insufficient  to  allow  of  analysis.  In  normal  urines 
no  such  residues  were  found. 

A  most  prolific  supply  of  alkaloids  from  the  urine  of  infectious 
diseases  has  been  furnished  by  Griffiths.  The  method  employed  was 
identical  with  that  described  by  Luff.  A  list  of  these  bases,  together 
with  others,  is  given  on  page  261. 

Hunter,  in  1890,  examined  the  urine  of  pernicious  anaemia  by  the 
benzoyl  chlorid  method,  and  obtained  a  very  small  quantity  of  a 
benzoyl  compound,  which  was  extremely  soluble  in  alcohol,  insoluble 
in  water.  It  crystallized  from  alcohol  in  long,  fine  needles,  grouped 
in  rosettes.  The  melting  point  was  at  174°-175*'.  The  crystalline 
form  and  the  melting  point  agreed  with  putrescin.  This  compound 
was  usually  alone,  but  sometimes  was  accompanied  by  another,  form- 
ing elongated,  rectangular  prisms.  The  crystalline  form  resembled 
that  of  the  cadaverin  compound.  One  specimen  of  urine  furnished  a 
dibenzoyl  compound,  crystallizing  in  long,  rectangular  prisms  having 
a  melting  point  between  70°  and  80°.  Binet  isolated  a  thermogenic 
substance  from  the  urine  of  tuberculosis,  and  to  a  less  extent  from 
normal  urine. 

Certain  basic  substances,  as  the  diamins,  cadaverin,  and  putrescin, 
have  been  isolated  in  a  perfectly  pure  condition.  These  two  basic 
substances  (see  page  265)  were  observed  by  Baumann  and  Udrdnzsky 
in  a  case  of  cystinuria.  Later,  Brieger  and  Stadthagen  demon- 
strated the  presence  of  these  same  bases  in  two  or  more  cases  of 
that  disease.  They  are  absent  in  normal  urine  and  feces,  and  ex- 
ceedingly rare  in  other  diseases.  Thus,  Roos  found  diamins  (putre- 
scin) in  only  one  case  of  cholera,  and  then  in  the  feces,  not  in  the 
urine  ;  more  frequently  in  diarrhoea  or  cholerine ;  also  in  one  case  of 
dysentery  and  malaria.  As  stated  above,  Hunter  apparently  suc- 
ceeded in  isolating  putrescin  from  the  urine  of  pernicious  ansemia. 

Cystin  and  the  diamins  are  now  known  to  be  proteid  cleavage 
products  and  their  presence  in  the  urine  is  more  easily  understood. 
The  hexon  bases  have  not  been  detected  in  urines  but  as  yet  no 
special  studies  have  been  made  in  that  direction.  The  presence  of 
ornithin  in  birds,  as  an  intermediate  waste  product,  was  recognized 
by  the  appearance  of  ornithuric  acid  in  the  urine. 

Poehl  has  proposed  the  following  method  for  the  estimation  of  the 
leucomains  in  the  urine.  To  100  c.c.  of  the  urine,  25  c.c.  of  hydro- 
chloric acid  (sp.  g.  1.134)  and  10  c.c.  of  a  10  per  cent,  solution  of 


468  CHEMISTRY  OF  THE  LEUCOMAINS. 

phosphotungstic  acid  are  added.  Albumin  and  pepton  must  first  be 
removed  if  present.  The  precipitate  is  allowed  to  subside  in  a 
graduated  tube,  and  the  number  of  cubic  centimeters  occupied  by  the 
precipitate  divided  by  8  is  to  represent  the  approximate  weight  of 
leucomains  per  liter  of  urine.  The  amount  thus  found  in  the  two 
cases  was  0.6  and  1.69.  Cavallero  and  Olivetti  have,  with  justice, 
severely  attacked  this  method,  and  have  shown  its  utter  unreli- 
ableness. 

It  is  now  a  well  established  fact  that  the  urine  of  certain  infectious 
diseases,  as  cholera  (Bouchard)  and  septicaemia  (Feltz),  etc.,  is  far 
more  poisonous  than  normal  urine.  That  the  poisons,  basic  or  other- 
wise, which  are  generated  within  the  body  by  the  activity  of  bacteria 
can  be  excreted  in  the  urine  is  seen  in  the  fact  that  immunity  to  the 
action  of  bacillus  pyocyaneus  has  been  conferred  on  animals  by  pre- 
vious injection  of  urine  taken  from  animals  inoculated  with  that 
bacillus  (Bouchard)  or  with  filtered  cultures  of  the  same  (Charrin 
and  Ruffer). 

Furthermore  the  excretion  of  the  tetanus  and  diphtheria  poisons 
by  the  urine  has  been  shown  to  take  place.  Thus,  Brunner  demon- 
strated the  tetanus  poison  in  the  urine  of  experimental  animals,  but 
failed  with  the  urine  of  the  disease  in  man.  Bruschettini,  however, 
with  the  urine  of  a  tetanus  patient,  produced  tetanic  symptoms  in 
mice  by  the  injection  of  3-10  c.c.  subcutaneously.  In  the  urine 
from  diphtheria  patients  Roux  and  Yersin  demonstrated  the  presence 
of  the  diphtheritic  poison  by  inducing  paralysis  in  animals.  Al- 
though basic  substances  are  not  present  in  the  urine  of  cholera,  they 
are  present,  but  less  frequently  than  was  expected,  in  the  discharges 
(putrescin  in  only  one  of  four  cases,  Roos).  From  cholera-feces 
Pouchet  extracted  an  oily  fluid  very  poisonous  to  frogs ;  whereas, 
Villi^rs  obtained  a  base  which  produced  convulsions  in  guinea-pigs. 
Kulneff  pointed  out  the  presence  of  ethylenediamin  (?)  in  the 
stomach-fluids  of  gastrectasia,  while  from  the  feces  of  a  case  of  gas- 
troptosis  he  isolated  trimethylamin. 

In  the  consideration  of  the  toxins  in  the  urine  of  infectious  dis- 
eases it  must  not  be  forgotten,  as  pointed  out  by  Jawein,  that  the 
poison  as  well  as  the  specific  germ  may  be  present  in  the  urine. 
Thus,  in  rabbits  that  died  as  a  result  of  infection  either  with  anthrax 
bacilli,  erysipelas  streptococci,  typhoid  bacilli,  or  with  pneumonia 
diplococci,  the  urine  was  found  to  contain  these  organisms.  It, 
therefore,  becomes  diflicult  to  decide  as  to  whether  the  toxin  is  elab- 
orated within  the  body  or  formed  subsequently  to  the  secretion  of 
the  urine. 

The  question  of  the  toxicity  of  normal  urine  has  been  the  subject 
of  considerable  controversy.  The  early  explanations  of  the  cause  of 
uraemia  assumed  that  urine  was  poisonous,  and  that  uraemic  symptoms 
were  the  result  of  the  retention  of  urine.     Actual  demonstrations  of 


LEUCOMAINS  OF  THE  URINE.  469 

the  toxicity  of  urine  were  made  early  in  the  century  by  Vauquelin 
and  others.  On  the  other  hand,  disbelievers  in  the  toxicity  of  urine 
were  not  wanting.  Thus  Frerichs  maintained  that  death,  resulting 
from  intravenous  injections  of  urine,  was  due  to  the  suspended  solid 
elements  of  the  urine  ;  that  urea  itself  was  harmless,  but  that  it  could 
by  the  action  of  a  ferment  give  rise  to  the  poisonous  ammonium  car- 
bonate. Voit  was  among  the  first  to  point  out  that  potassium  salts, 
on  account  of  their  toxicity,  could  play  an  important  part  in  uraemia. 
It  can  now  be  positively  stated  that  normal  urine  does  possess  a  cer- 
tain degree  of  toxicity.  It  is  more  difficult  to  decide  upon  the  nature 
of  this  poison.  Feltz  and  Ritter  (1881),  and  independently  Asta- 
schewsky,  arrived  at  the  opinion  that  the  toxicity  was  chiefly  due  to 
the  potassium  salts  of  the  urine.  Schiffer,  while  acknowledging  the 
presence  and  action  of  the  inorganic  salts,  maintained  that  the  urine 
contained  a  definite  organic  poison,  for  the  reason  that  the  concen- 
trated aqueous  solutions  from  alcoholic  extracts  of  the  urine  residue, 
deprived  of  inorganic  salts,  killed  large  rabbits  in  doses  correspond- 
ing to  1-1 1  liters  of  urine. 

According  to  Bouchard,  30-60  c.c.  of  normal  urine,  injected  in- 
travenously, will  kill  a  rabbit  weighing  one  kilogram.  Hence  a 
man  weighing  60  kilograms,  and  excreting  per  day  1200  c.c,  would, 
if  50  c.c.  are  necessary  to  kill  one  kilogram  of  living  matter,  secrete 
enough  poison  to  kill  twenty-four  kilograms  of  animal.  Inasmuch 
as  the  amount  necessary  to  kill  one  kilogram  of  animal  is  designated 
as  one  urotoxy,  therefore,  in  the  above  case  twenty-four  urotoxies 
are  formed  per  day.  The  urotoxic  coefficient  is  the  number  of  uro- 
toxies which  one  kilogram  of  man  forms  in  twenty-four  hours. 
Therefore,  |4-  =  0.4,  the  urotoxic  coefficient.  The  average  normal 
urotoxic  coefficient  is  placed  by  Bouchard  at  0.464.  It  follows, 
therefore,  that  an  average  man  would,  if  the  excretion  of  urine  was 
stopped,  be  killed  in  fifty-two  hours.  The  variations  of  the  urotoxic 
coefficient  in  the  normal  individual  is  limited.  In  disease  it  rarely 
exceeds  2,  and  rarely  falls  below  0.10. 

According  to  Bouchard,  five  kinds  of  poisons  may  be  met  with  in 
the  urine,  producing  narcosis,  salivation,  mydriasis,  paralysis,  and 
convulsions.  The  day  urine,  which  is  chiefly  narcotic,  is  2-4  times 
more  toxic  than  the  sleep  urine,  which  induces  convulsions  and  is 
antagonistic  to  the  former.  The  toxicity  is  independent  of  the  den- 
sity, since  night  urine  is  more  dense  than  that  secreted  during  the  day. 

The  greater  part  of  the  toxicity  of  urine  is  ascribed  by  Bouchard  to 
organic  poisons,  especially  coloring-matters,  whereas  potassium  salts 
are  regarded  as  the  cause  of  but  a  small  fraction  of  the  toxicity. 

Lepine  likewise  found  that  about  60  c.c.  of  urine  sufficed  to  kill 
one  kg.  of  animal.  The  inorganic  salts,  however,  were  ascribed  by 
him  a  much  greater  importance,  inasmuch  as  he  estimated  that  85 
per  cent,  of  the  intoxication  was  due  to  this  cause.     The  remainder 


470  CHEMISTRY  OF  THE  LEUCOMAINS. 

of  the  toxicity  was  due  to  organic  matter.  Stadthagen  arrived  at 
practically  the  same  results,  that  80-85  per  cent,  of  the  toxicity  was 
due  to  the  inorganic  constituents.  A  part  of  the  toxicity,  15-20  per 
cent.,  is  therefore  due  to  organic  substances.  No  one  organic  sub- 
stance in  the  urine,  as  urea,  creatin,  etc.,  possesses  this  toxicity. 
Stadthagen  has  further  shown  that  alkaloidal  bodies  as  peptotoxin, 
guanidin,  methyl  guanadin,  cholin,  neurin,  xanthocreatinin  are  ab- 
sent from  normal  urine.  100  liters  of  urine  examined  by  Brieger's 
method  for  bases  gave  only  ammonia,  a  little  trimethylamin,  besides 
creatinin.  Dresbach^  (1900),  employing  the  same  method,  obtained 
poisonous  extracts.  Gautier  has  supposed  that  the  urine  poison  was 
a  proteid  analogous  to  that  in  the  venom  of  serpents,  but  Stadthagen 
showed  that  proteids  were  absent.  Ferments  like  pepsin  were  also 
excluded  from  consideration  because  of  their  minute  amount.  His 
experiments  were  largely  negative,  so  far  as  the  detection  of  an 
organic  poison  was  concerned.  Stadthagen  disproved  the  existence 
of  a  special  urine  poison.  The  poisonous  action  of  normal  urine  is 
therefore  due  to  the  sum  total  action  of  the  inorganic  salts,  chiefly 
potassium,  and  the  normal  organic  constituents  as  urea,  creatinin, 
etc.,  which  by  themselves  possess  but  slight  action. 

Guinard  recently  tested  the  action  of  normal  urine  from  dif- 
ferent animals.  On  an  average,  the  toxicity  per  kg.  rabbit  was  as 
follows :  Dog,  193  c.c. ;  man,  132.7  c.c. ;  pig,  53  c.c. ;  ox,  38.5 
c.c. ;  guinea-pig,  35  c.c. ;  sheep,  33.8  c.c.  ;  goat,  32  c.c. ;  ass,  29.4 
c.c. ;  horse,  29.2  c.c. ;  rabbit,  16  c.c. ;  cat,  13  c.c.  The  urine  of  a 
bear  possessed  toxicity  similar  to  that  of  the  dog ;  that  of  the  lion 
and  tiger  corresponded  to  that  of  the  cat.  In  the  case  of  the  horse 
the  urine  was  less  toxic  from  weak  animals,  from  young  animals,  and 
from  males  than  from  strong  or  old  animals  or  females.  The  urea 
per  liter  of  urine  varied  from  15  g.  in  the  dog  to  118  g.  in  the  cat. 
While  rabbits  are  killed,  per  kilo,  by  an  injection  of  45  c.c.  of  nor- 
mal urine,  dogs  are  killed  by  an  intravenous  injection  of  100  c.c.  per 
kilo  (Mairet  and  Bosc).  If  the  thyroid  gland  is  removed,  the  toxic 
effects  are  increased  (Godart  and  Slosse). 

While  Guinard  failed  to  observe  any  effect  or  toxicity  following 
the  injection  of  the  urine  of  pregnancy,  Chambrelent  and  Demont 
found  that  the  toxicity  was  diminished  in  the  later  months.  The 
average  urotoxic  coefficienjb  was  0.27.  Mairet  and  Bosc  examined 
the  toxicity  of  the  urine  in  nervous  disorders  and  found  it  to  be  in- 
creased, especially  in  lypemania  and  mania.  In  general,  however, 
the  toxic  action  was  the  same  as  that  of  normal  urine,  though  at 
times  it  produced  specific  nervous  symptoms  approximating  those  of 
the  disease. 

Increased  toxicity  of  the  urine  was  observed  by  Surmont  in 
atrophic  cirrhosis,  tuberculosis,  and  carcinoma  of  the  liver.  On  the 
^Journ.  Exp.  Med.,  5,  315,  1900. 


LEUCO MAINS  FROM  OTHER  TISSUES  OF  THE  BODY.      471 

other  hand,  the  toxicity  was  normal  or  subnormal  in   hypertrophic 
cirrhosis,  in  hepatic  congestion,  and  in  infectious  icterus. 

Roque  and  Lemoine  showed  that  there  are  marked  changes  in  the 
toxicity  of  the  urine  in  malaria  before  and  after  an  attack.  Before 
an  attack  the  urotoxic  coefficient  was  0.13  and  0.274,  whereas  after 
an  attack  it  rose  to  0.684  and  1.276.  It  would  appear,  therefore, 
that  toxic  products  result  from  the  growth  of  the  malarial  plasmodium 
in  the  blood,  and  are  largely  eliminated  by  the  kidneys.  Quinin 
favors  this  excretion  of  poisons. 

It  does  not  follow  from  what  has  been  stated  that  the  urine  in  dis- 
ease is  always  more  poisonous  than  in  health.  There  are  diseases,  as 
uraemia,  where,  as  shown  by  Schiffer  and  Bouchard,  the  urine  is  less 
toxic  than  in  health.  This  may  be  due  to  a  retention  of  the  salts  of 
potassium. 

Leucomams  of  the  Saliva. 

According  to  Gautier  (1881),  normal  human  saliva  contains  divers 
toxic  substances  in  small  quantities  which  differ  very  much  in  their 
action  according  to  the  time  of  their  secretion,  and  probably  accord- 
ing to  the  individual  gland  in  which  they  are  secreted.  The  aqueous 
extract  of  saliva  at  100°  is  poisonous  or  narcotic  in  its  action 
toward  birds.  To  show  the  presence  of  basic  substances,  the  aqueous 
extract  was  slightly  acidulated  with  dilute  hydrochloric  acid,  then 
precipitated  by  Mayer's  reagent;  the  precipitate  was  washed,  then 
decomposed  by  hydrogen  sulphid,  and  the  solution  filtered.  The 
filtrate  on  evaporation  gave  a  residue  consisting  of  microscopic 
slender  needles  of  a  soluble  hydrochlorid.  This  salt,  purified  by 
extraction  with  absolute  alcohol,  formed  soluble,  crystalline,  but 
easily  decomposable  double  salts  with  platinum  chlorid  and  with 
gold  chlorid.  The  solution  of  the  hydrochlorid  produced  an  im- 
mediate precipitate  of  Prussian  blue  in  a  mixture  of  potassium 
ferricyanid  and  ferric  chlorid,  and  when  injected  into  birds  produced 
stupor. 

Leucomains  from  Other  Tissues  of  the  Body. 

Selmi's  work  upon  the  formation  of  ptomains  during  the  process 
of  putrefaction  led  many  investigators  to  doubt  the  production  of 
these  bases  by  the  decomposition  of  the  proteid  or  other  complex 
molecules.  To  substantiate  this,  a  number  of  chemists,  especially 
Italian,  endeavored  to  show  that  Selmi's  bases,  to  a  large  extent  at 
least,  exist  preformed  in  the  various  tissues.  Paterno  and  Spica 
(1882)  succeeded  in  extracting  from  fresh  blood  as  well  as  from  fresh 
albumin  of  eggs  substances  identical,  or  at  least  similar,  to  those 
designated  under  the  name  of  ptomains.  Their  observations,  how- 
ever, were  confined  to  the  detection  of  alkaloidal  reactions  in  the 
various  extracts  obtained  by  Dragendorff's  method,  and  at  no  time 
were  they  in  possession  of  a  definite  chemical  individual.     Marino- 


472  CHEMISTRY  OF  THE  LEUCOMAINS. 

Zuco  (1885)  was  more  successful,  inasmuch  as  he  succeeded  in  ob- 
taining from  fresh  tissues  and  organs  relevant  quantities  of  a  base 
identical  with  cholin,  and,  in  addition,  he  obtained  extremely  minute 
traces  of  other  alkaloidal  bodies.  One  of  these,  obtained  by  the  Stas 
method  from  the  liver  and  spleen  of  an  ox,  exhibited  in  hydrochloric 
acid  solution  a  beautiful  violet  fluorescence  resembling  very  much 
that  of  the  salts  of  quinin.  A  similar  base,  probably  identical  with 
this  one,  was  obtained  by  Bence  Jones  and  Dupre  (1856)  from  liver, 
nerves,  tissues,  and  other  organs,  and  was  named  by  them  "  animal 
chinoidin."  A  greenish-blue  fluorescence  is  frequently  observable 
in  the  alcoholic  extracts  of  decomposing  glue  as  well  as  from  other 
putrefying  substances,  and  is  undoubtedly  due  to  products  formed 
by  some  one  of  the  fluorescing  bacteria.  From  a  number  of  very 
thorough  experiments,  he  concluded  that  basic  substances  do  not 
preexist  in  fresh  organs,  but  that  the  acids  employed  in  the  process 
of  extraction  exert  a  decomposing  action  upon  the  lecithin  present  in 
the  tissues,  resulting  in  the  formation  of  cholin.  He  further  showed 
that  the  method  of  Dragendorff,  on  account  of  the  larger  quantity  of 
extractives,  which  forms  invariably  gave  a  larger  yield  of  this  base 
than  did  the  Stas-Otto  method.  Similar  observations  were  made  by 
Guareschi  and  Mosso,  by  Coppola  and  others.  At  the  present  time 
there  is  no  doubt  that  some  basic  substances,  among  these  cholin  and 
the  hexon  bases,  can  be  formed  by  the  action  of  reagents,  and,  on 
the  other  hand,  it  is  equally  well  demonstrated  that  similar  bases  do 
preexist  in  the  physiological  condition  of  the  tissues  and  fluids  of  the 
body. 

Recently  R.  Wurtz  has  obtained  from  normal  blood  a  number  of 
crystalline  products  of  alkaline  reaction,  which  form  well  crystalliz- 
able  double  salts  with  gold,  platinum,  and  mercuric  chlorids.  These, 
however,  have  not  been  as  yet  subjected  to  analysis,  because  of  the 
minute  quantities  which  were  isolated. 

Marino-Zuco  and  Martin  in  1894  showed  the  presence  of  cholin 
in  fresh  blood. 

In  1899  Gulewitsch  carried  out  a  most  painstaking  investigation 
on  the  leucomains  in  fresh  brains  with  especial  reference  to  the  de- 
tection of  the  poisonous  neurin.  The  possible  presence  of  the  latter 
has  been  suggested  as  explaining  the  auto-intoxications  met  with  in 
mental  disorders.  By  means  of  Brieger's  method  he  obtained  cholin 
but  no  neurin.  Moreover,  he  was  unable  to  obtain  the  latter  from 
protagon.  The  aqueous  brain  extract  gave  a  very  small  quantity  of 
two  bases,  probably  diamins.     Urea  was  also  obtained. 

In  extensive  skin-burns  Kijanitzin  isolated  a  peptotoxin-like  base 
from  the  urine  and  blood,  more  abundantly  from  the  organs.  A 
similar  base  was  shown  by  him  to  be  produced  by  the  action  of  gastric 
juice  on  the  blood  in  the  presence  of  bacteria;  also  in  the  early  stages 
of  the  decomposition  of  blood.     The  explanation  of  the  fatal  results  fol- 


LEUCOMAINS  FROM  OTHER   TISSUES  OF  THE  BODY.      473 

lowing  the  varnishing  of  a  part  or  the  whole  of  the  body  is  given  on  pp. 
329,  466.    A  similar  explanation  undoubtedly  holds  true  for  uraemia. 

The  presence  of  specific  toxic  substances  in  the  blood  of  infectious 
diseases  is  well  recognized.  Nissen  has  shown  that  the  blood  in  sup- 
puration was  toxic.  In  the  blood  of  tetanus  in  man  Kallmeyer  and 
Nissen  demonstrated  the  presence  of  the  tetanic  poison.  Immerwahr 
showed  the  same  to  be  true  with  the  organs  of  experimental  tetanic 
animals,  and  that  the  blood  of  scarlet  fever  during  uraemia  was  toxic. 
Brieger  was  the  first  to  show  the  presence  of  tetanin  in  the  amputated 
arm  of  a  patient. 

Morelle  (1886)  showed  the  presence,  in  the  spleen  of  the  ox,  of  a 
base,  the  hydrochlorid  of  which  crystallized  in  deliquescent  needles 
and  likewise  formed  crystalline  platino-  and  aurochlorids.  From  ex- 
periments made  by  Laborde,  the  base  would  seem  to  possess  decided 
toxic  properties,  bringing  on  a  dyapnoeic  condition  with  convulsive 
movements  and  loss  of  motion.  The  post-mortem  examinations  re- 
vealed an  extended  visceral  oedematous  infiltration,  and  stoppage  of 
the  heart  in  systole.  For  the  presence  of  the  xanthin  bases,  cystin, 
gerontin,  etc.,  in  the  organs  of  the  body,  see  preceding  pages. 

Viron  found  a  very  poisonous  albuminoid  in  a  hydrocele  fluid  from 
a  sheep. 

A.  W.  Blyth  claimed  to  have  isolated  from  milk  two  alkaloidal 
substances,  namely,  galactin,  the  lead  salt  of  which  is  said  to  have 
the  formula  PbgOj.Cg^HjgN^Ojj ,  and  lactochrome,  the  mercury  salt  of 
which  is  represented  by  the  formula  HgO.CgHjgNOg. 

The  adrenals  have  been  the  subject  of  repeated  investigations  dur- 
ing the  past  half  a  century,  but  really  valuable  results  have  been  ob- 
tained only  within  the  last  three  or  four  years.  At  an  early  date  the 
fact  was  established  that  these  organs  perform  an  extremely  impor- 
tant function  inasmuch  as  their  extirpation  is  promptly  followed  by 
death.  The  rapid  and  severe  effects  observed  have  been  generally 
regarded  as  due  to  an  intoxication  of  the  organism  with  products 
which  normally  were  supposed  to  be  destroyed  in  the  adrenals. 
Thus  Marino-Zuco  obtained  cholin  from  the  adrenals  and  moreover 
with  Dutto  he  obtained  this  base  in  the  urine  of  Addison's  disease. 
These  investigators  and  also  Carbone  obtained  results  showing  that 
cholin  was  decidedly  more  toxic  to  animals  deprived  of  the  adrenals 
than  to  normal  animals.  Furthermore,  the  blood  of  animals  from 
which  the  adrenals  were  removed  has  been  shown  to  possess  a 
marked  toxic  action.  Only  recently,  Levin  ^  has  shown  that  the 
blood  of  such  animals  contains  something  which  acts  on  the  blood 
pressure  and  which  does  not  exist  in  normal  blood. 

The  early  studies  of  Oliver  and  Schafer  and  others  demonstrated 
that  the  adrenals  contain  a  substance  which  exerts  a  marked  action 
upon  the  blood  pressure,  and  thus  led  to  a  careful  study  and  search 
^  Amer,  Journ.  Physiology,  5,  358,  1901. 


474  CHEMISTRY  OF  THE  LEUCOMAINS. 

for  the  active  principle.  According  to  Miihlmann  this  substance  is 
pyrocatechin.  Fiirth  ^  at  first  also  held  that  the  active  principle  was 
a  pyrocatechin-like  body,  but  eventually  he  considered  it  to  be  a 
hydro-dioxypyridin  having  the  formula  CgHyNOj  or  CgHgNOg .  To 
this  substance  be  applied  the  name  suprarenin.  In  this  country 
most  painstaking  investigations  were  made  by  AbeP  who  succeeded 
in  isolating  a  basic  product  which  he  designated  as  epinephrin. 
The  formula  which  he  deduced,  Cj^^H^gNO^,  is  not  that  of  the 
free  base  but  rather  that  of  a  mono-benzoyl  derivative.  Accord- 
ing to  Takamine  ^  the  blood  pressure  raising  principle  of  the  gland 
can  be  isolated  in  a  pure  crystalline  form.  He  has  designated  this 
product  as  adrenalin  and  to  this  Aldrich^  ascribed  the  formula 
CjHjgNOg.  Abel  however  has  shown  that  the  analytical  results 
agree  more  closely  with  the  formula  C^oHj^NOg  which  he  had  pro- 
posed. It  appears  from  Abel's  recent  work  that  adrenalin  does  not 
exist  as  such  in  the  glands  but  is  rather  a  modified  product  of 
epinephrin,  the  active  base. 

It  is  evident  from  this  that  the  nature  of  the  active  constituent  of 
the  suprarenals  is  as  yet  undetermined  and  for  this  reason  it  is  hardly 
necessary  to  enter  into  a  detailed  consideration  of  its  properties. 

The  purin  bases  in  adrenals  have  been  studied  by  Oberblom 
(p.  390).  The  perfectly  fresh  glands  yielded  a  smaller  quantity 
of  these  bases  than  such  as  were  digested  for  two  days  at  the  body 
temperature  in  the  presence  of  chloroform.  Assuming  that  the  ac- 
tion of  bacteria  was  wholly  eliminated  it  would  seem  that  this  in- 
crease in  bases  was  probably  due  to  the  action  of  an  enzyme.  Xan- 
thin,  1-methyl  xanthin,  hypoxanthin,  epiguanin  and  adenin  were 
detected.  The  presence  of  methyl  xanthin  and  methyl  guanin  is  of 
interest  in  view  of  the  fact  that  these  derivatives  have  been  looked 
upon  as  cleavage  products  derived  from  the  food.  If  Oberblom's 
results  can  be  confirmed  it  would  indicate  the  existence  of  a  purin 
antecedent  as  for  example  a  1.7-dimethyl  guanin  which  in  the  body 
might  give  rise  to  paraxanthin  or  like  the  latter  might  be  demethyl- 
ated,  forming  the  1-methyl  and  7-methyl  compounds  referred  to 
above. 

Venoms  of  Poisonous  Serpents. 

The  study  of  the  chemistry  of  the  venoms  of  serpents  and  of 
batrachians  is  fraught  with  so  many  difficulties  and  with  so  much 
danger,  that  we  cannot  wonder  at  the  present  unsatisfactory  con- 
dition of  our  knowledge  in  regard  to  the  poisonous  principles  which 
they  contain.  Much  of  the  early  work  was  not  only  inaccurate  and 
very  contradictory,  but  was  far  from  meeting  the  requirements  of 

^Zdt.  physiol.  Chem.,  24,  142  ;  26,  15  ;  29,  105. 

^Zeit.  phygiol.  Chem.,  28,  318;  Johns  HopkinsHosp.  Bull,  1901,  84,  337;  1902,  29. 

'^Journ.  Am.  Med.  Ashoc,  38,  150,  153. 

*  Am^r.  Joum.  Physiology,  5,  457  (1901). 


VENOMS  OF  POISONOUS  SERPENTS.  475 

exact  toxicological  research.  Recent  investigations,  however,  have 
made  it  certain  that  the  most  active  constituent  of  the  venom  of 
serpents  is  not  alkaloidal  in  its  nature,  as  was  supposed  by  some. 
In  1881  Gautier  announced  the  isolation  of  two  alkaloids  from  the 
venom  of  the  cobra  which  gave  precipitates  with  tannin,  Mayer's 
reagent,  Nessler's  reagent,  iodin  in  potassium  iodid,  etc.  They 
formed  crystallizable  platinochlorids  and  aurochlorids,  and  also  crys- 
talline, neutral,  somewhat  deliquescent  hydrochlorids.  The  neutral 
or  slightly  acid  solutions  produced  an  immediate  precipitate  of 
Prussian  blue  in  a  mixture  of  potassium  ferricyanid  and  ferric 
chlorid.  The  substances  possessed  a  decided  physiological  action, 
but  Gautier  himself  did  not  consider  them  to  be  the  most  dangerous 
constituents  of  the  venom.  This  observation  of  Gautier  as  to  the 
presence  of  distinct  basic  substances  in  venoms  is  at  variance  with 
that  of  Wolcott  Gibbs,  who  was  unable  to  obtain  an  alkaloid  from 
the  rattlesnake  (Crotalus)  venom.  S.  .Weir  Mitchell  and  E.  T. 
Reichert  were  likewise  unable  to  substantiate  Gautier's  statements. 
Subsequently,  Wolfendeu,  in  an  elaborate  paper  on  the  nature  of 
cobra  venom,  confirmed  Wolcott  Gibbs  as  to  the  entire  absence  of 
any  alkaloidal  body.  , 

Mitchell  and  Reichert  made  a  careful  study  of  the  venoms  of 
various  serpents,  such  as  cobra,  rattlesnake,  moccasin,  and  Indian 
viper,  and  succeeded  in  isolating  two  proteid  constituents,  one 
belonging  to  the  class  of  globulins  and  the  other  to  the  peptons. 
The  pepton  is  said  to  be  non-precipitable  by  alcohol.  According  to 
them,  the  globulin  constituent  consisted  of  at  least  three  distinct 
globulins.  They  found  that  boiling  coagulated  and  destroyed  the 
globulin  as  a  poison,  but  that  the  venom  pepton  was  toxically  un- 
changed, so  that  the  solution,  though  still  poisonous,  failed  to  pro- 
duce the  characteristic  local  lesions  due  to  fresh  or  unboiled  venom. 
On  the  other  hand,  Gautier  asserted  that  the  venom  was  not  sensibly 
altered  on  being  heated  to  120°- 125°  and  that  the  toxic  action 
remained  constant  even  when  all  the  proteid  constituents  were  re- 
moved, thus  showing  that  the  toxic  action  cannot  be  attributed  to  the 
albuminoids.  Later,  Quartier  acknowledged  that  viper  venom  was 
destroyed  at  100°.  Calmette  found  it  to  be  destroyed  at  98°,  while 
still  later  Phisalix  and  Bertrand  showed  that  an  exposure  of  five 
minutes  at  80°- 85°  destroyed  the  toxicity.  The  venom  pepton 
from  the  rattlesnake  or  the  moccasin,  however,  when  injected  into 
animals  produced  toxic  effects  which  were  marked  by  an  oedematous 
swelling  over  the  site  of  injection ;  the  tumor  was  filled  with  serum, 
and  so  also  was  the  subcutaneous  cellular  tissue.  Furthermore, 
a  gradual  breaking  down  of  the  tissues  occurred,  accompanied  by 
rapid  putrefactive  changes  and  a  more  or  less  extensive  slough. 
That  peptons  may  possess  intensely  poisonous  properties  has  been 
shown  to  be  the  case  by  a  number  of  authors,  among  whom  may  be 


476  CHEMISTRY  OF  THE  LEUCOMAINS. 

mentioned  Schniidt-Miilheim,  Hofmeister,  Pollitzer,  and  others. 
Brieger  believed  that  the  formation  of  peptons  in  the  process  of 
digestion  was  accompanied  by  the  development  of  a  toxic  ptomain, 
which  he  named  peptotoxin.  As  stated  elsewhere,  Salkowski  has 
very  properly  questioned  the  formation  of  peptoxin  in  the  ordinary 
digestion  of  proteids. 

The  venom  globulins,  on  the  other  hand,  though  present  in  less 
quantity  than  the  peptons,  induced  the  same  remarkable  local  effects 
seen  on  injection  of  the  pure  venom.  They  caused  local  bleedings, 
destroyed  the  coagulability  of  the  blood,  and  rapidly  corroded  the 
capillaries. 

These  results  of  Mitchell  and  Reichert,  which  are  given  somewhat 
in  full,  have  been  questioned  by  Wolfenden,  who,  while  agreeing  in 
the  main  that  the  poisonous  property  of  venom  is  due  to  proteid 
constituents,  regarded  their  pepton  not  as  a  true  pepton,  but  rather 
as  one  or  more  bodies  of  the  albumose  group  of  proteids.  He  like- 
wise regards  the  globulin  of  moccasin  venom  to  be  some  other 
proteid  body.  According  to  him,  the  cobra  venom  owed  its  toxicity 
to  the  proteids,  globulin,  serum  albumin,  and  acid  albumin.  Occa- 
sionally there  seem  to  be  present  traces  of  pepton  and  of  hemial- 
bumose. 

Brieger  was  at  first  apparently  inclined  to  believe  that  the  action 
of  venom  was  due  to  animal  alkaloids,  on  the  ground  that  these 
bases  are  extremely  soluble,  and  hence  always  go  into  solution,  along 
with  the  likewise  very  soluble  proteid  constituents,  and  that  the 
difficulty  in  their  isolation  lies  in  the  elimination  of  these  proteids. 
Subsequently,  however,  Brieger  and  Frankel  pointed  out  the 
poisonous  nature  of  some  bacterial  proteids  (toxalbumins)  and  also 
showed  that  cobra  poison  yields  with  alcohol  a  precipitate  which 
gives  proteid  reactions. 

The  proteids  of  serpents'  venom  should  be  compared  with  the 
toxins  formed  by  the  activity  of  the  pathogenic  bacteria,  and  also 
with  the  similar  compounds,  the  phytalbumoses  of  castor  beans, 
jequirity,  etc.,  and  with  the  enzymes.  Possibly  similar  compounds 
will  be  found  in  croton  and  other  species  of  ricinus,  jatropha,  loco- 
weed,  etc.  The  poisons  secreted  by  certain  spiders  and  fish  may  be 
mentioned  in  this  connection  (see  p.  193). 

Recent  researches  on  the  venoms  have  been  productive  of  very 
important  results,  especially  from  the  standpoint  of  immunity  to  and 
cure  from  the  bites  of  venomous  serpents.  Although  in  the  higher 
latitudes  poisoning  from  snake-bites  is  comparatively  rare,  it  should 
not  be  overlooked  that  in  certain  portions  of  the  globe,  notably 
India  and  Australia,  the  mortality  from  this  cause  is  exceedingly 
high,  and  may  well  claim  the  attention  of  governments.  Thus,  it  is 
estimated  in  India  that  over  20,000  persons  die  annually  from  the 
bites  of  serpents. 


VENOMS  OF  POISONOUS  SEItPENTS.  477 

Phisalix  and  Bertrand  in  1893  confirmed  Fontana's  previous 
observation  that  the  garter-snake  (couleuvre)  was  unaffected  by 
repeated  bites  from  vipers  or  by  subcutaneous  injection  of  the 
venom  of  the  viper.  These  authors  showed  that  a  dose  of  the 
venom  sufficient  to  kill  15  to  20  guinea-pigs  was  without  effect 
on  the  garter-snake.  The  natural  immunity  of  the  garter-snake 
to  the  viper  venom  is  thus  firmly  established.  From  previous 
researches  on  the  natural  immunity  of  the  "  crepaud  "  and 
viper  to  their  own  venoms  Phisalix  and  Bertrand  showed  that  the 
blood  or  serum  of  these  serpents  contained  the  same  poison,  echidnin, 
as  was  present  in  the  venom.  Similarly  the  blood  or  serum  of  the 
garter-snake  when  injected  in  doses  of  1.5  c.c.  intraperitoneally  into 
guinea-pigs  produced  death  in  two  hours  with  the  same  symptoms 
as  are  observed  after  poisoning  with  viper  venom.  Although  the 
several  forms  of  garter-snakes  are  considered  as  non-venomous,  they 
nevertheless  secrete  through  the  superior  maxillary  gland  (or  special 
glands,  Jourdain)  toxic  products  analogous  to  echidnin,  which  are 
excreted  into  the  blood,  rendering  this,  therefore,  highly  poisonous, 
and  at  the  same  time  establishing  natural  immunity.  After  the 
ablation  of  the  venom  glands  in  the  viper  the  blood  loses  a  part  of 
its  toxicity,  showing  that  the  source  of  the  poison  in  the  blood  is  the 
venom  gland.  It  Avould  seem  that  this  immunity  is  one  of  tolerance 
and  analogous  to  that  which  Sewall  obtained  with  rattlesnake  venom. 

Later  (February,  1894),  Phisalix  and  Bertrand  showed  that  viper 
venom  heated  to  75°— 85°  for  five  minutes  lost  its  poisonous  property 
with  respect  to  guinea-pigs  and  acted  as  a  vaccine.  The  temperature 
of  animals,  however,  was  raised,  whereas  with  unheated  venom  it  is 
lowered.  They  were,  therefore,  led  to  believe  that  viper  venom 
contained  (1)  a  phlogogenic  substance  like  the  diastases — echidnase, 
and  (2)  a  general  poison — echidnotoxin.  Since  both  are  destroyed  by 
heat  the  vaccine  results  either  from  the  destruction  of  these  two  sub- 
stances or  is  preformed  in  the  venom  and  acts  after  the  toxic  prin- 
ciples are  destroyed.  This  behavior  of  venom  to  heat  and  to  the 
production  of  immunity  is  analogous  to  Frankel's  method  of  produc- 
ing immunity  to  diphtheria. 

The  heated  viper  venom,  or  vaccine,  does  not  impart  immediate 
immunity  to  guinea-pigs,  but  this  condition  follows  after  the  lapse  of 
several  days — a  result  of  the  reaction  of  the  organism.  An  anti- 
toxin appeared  in  the  blood  after  the  injection  of  the  echidnovaccine, 
and  in  less  amount  in  the  blood  when  immunity  has  been  estab- 
lished by  tolerance.  The  amount  of  antitoxin  in  the  blood  could  be 
increased  as  in  the  case  of  tetanus  and  of  diphtheria.  A  very  short 
time  afterward  Calmette  confirmed  the  observations  that  animals 
could  be  immunized  by  repeated  injections  of  the  venom,  beginning 
in  small  doses  and  gradually  increasing.  A  single  non-fatal  injec- 
tion of  venom  may  produce  antitoxin  in  the  blood  of  the  animal. 


478  CHEMISTRY  OF  THE  LEUCOMAINS. 

He  furthermore  obtained  immunity  by  applying  the  method  em- 
ployed by  Roux  and  Vaillard  in  their  work  on  tetanus,  that  is,  by 
repeated  injections  of  the  venom  mixed  with  gold  chlorid,  or  sodium 
or  calcium  hypochlorite.  The  serum  of  the  immunized  animal  was 
found  to  be  antitoxic  in  the  same  sense  as  the  serum  of  animals  im- 
munized to  diphtheria  or  tetanus.  Furthermore,  not  only  was  the 
blood  shown  to  be  antitoxic  to  the  venom  employed,  but  also  to  the 
venoms  of  other  serpents.  Thus,  the  serum  of  a  rabbit  immunized 
against  the  cobra  venom  is  not  only  antitoxic  to  this  venom,  but  also 
to  the  viper  of  France,  the  black  snake  of  Australia,  etc. 

Immunity,  therefore,  to  venom  can  be  obtained  (1)  by  repeated 
injections  of  small  doses  (Sewall,  Phisalix  and  Bertrand,  Calmette); 

(2)  by  the  use  of  heated  venom  or  vaccine  (Phisalix  and  Bertrand); 

(3)  by  repeated  injections  of  venom  mixed  with  hypochlorite  solution 
(Calmette);  (4)  by  injections  of  antitoxic  serum  (Phisalix  and  Ber- 
trand, Calmette).  The  immunity  according  to  the  first  method,  by 
tolerance,  has  been  shown  to  be  due  to  the  presence  of  antitoxin  sub- 
stances in  the  blood  (Phisalix  and  Bertrand,  Calmette,  Fraser). 
The  second  and  third  methods  are  explainable  in  the  same  way. 

The  application  of  the  latter  principle  in  the  treatment  of  bites 
from  serpents  was  suggested  by  Phisalix  and  Bertrand  and  carried 
out  by  Calmette.  The  rabbit,  dog  (?),  guinea-pig  (Phisalix  and 
Bertrand),  horse  and  ass  have  been  employed  to  furnish  serum  anti- 
toxic to  venom.  Calmette  has  prepared  a  serum  of  a  strength  of 
1  :  10,00D;  that  is,  rabbits  given  a  dose  of  venom  sufficient  to  kill 
in  three  or  four  hours  are  saved  if  a  quantity  of  antitoxic  serum  cor- 
responding to  y-g-^o^-g-  of  their  weight  is  injected  not  later  than  one 
hour  after  the  injection  of  venom.  As  stated  above,  this  antitoxic 
serum  protects  against  all  venoms. 

Calmette  has  also  shown  that  the  ichneumon  of  the  Antilles  is 
naturally  immune  to  venom,  and  that  it  owes  this  condition  to  the 
antitoxic  property  of  its  blood.  Just  as  the  serum  of  man  or  of  the 
horse  was  sometimes  found  to  be  antitoxic  to  the  diphtheria  poison, 
so  the  serum  of  dogs  was  occasionally  found  to  be  antitoxic  to 
venom. 

The  studies  of  Phisalix  *  and  others  show  that  protection  against 
viper  venom  may  be  secured  by  the  injection  of  serum  of  diverse 
origin.  The  serum  of  the  vipers,  garter-snakes,  eels,  hedge-hog, 
guinea-pig,  frog,  toad,  dog  and  horse  possesses  such  action.  More- 
over, Fraser  has  found  that  minute  quantities  of  bile  possess  a  like 
action.  Phisalix  has  not  only  confirmed  this  observation  but  has  , 
demonstrated  that  the  bile  acids,  cholesterin  and  tyrosin  vaccinate 
against  viper  venom.  The  protecting  property  however  is  lost  when 
the  material  is  heated  to  120°.     The  alcoholic  precipitates  produced 

1  Gmipt.  Bendus,  121,  745  ;  122,  1439  ;  123,  1305  ;  125,  121,  977,  1053  ;  127, 
1036  ;  126,  431. 


VENOMS  OF  POISONOUS  SERPENTS.  479 

in  extracts  of  dog's  liver  or  pancreas  also  protect,  Phisalix  has  also 
found  that  extracts  of  mushrooms  (Agaricus  edulis)  and  the  venom 
of  hornets  exert  a  similar  protective  action.  These  various  sub- 
stances are  not  strictly  speaking  antitoxins.  They,  like  porcelain, 
merely  modify  the  venom.  This  may  be  due  to  changes  analogous 
to  surface  action. 

Fraser  has  independently  arrived  at  substantially  the  same  results 
as  the  French  investigators.  Serum  that  is  antitoxic  to  venom  is 
designated  by  Fraser  as  antivenin.  He  immunized  the  horse  and 
cat  against  the  cobra  venom.  The  cat  was  also  rendered  immune  by 
administration  through  the  stomach.  It  is  interesting  to  note  that 
Repin  obtained  immunity  in  guinea-pigs  to  abrin,  the  poisonous  albu- 
mose  of  jequirity,  by  repeated  administrations  of  small  doses  by  the 
mouth.  Ehrlich  has  shown  that  while  -^^  mg.  of  abrin  is  sufficient 
to  kill  a  guinea-pig  in  two  or  three  days  when  injected  subcutane- 
ously,  one  hundred  times  this  amount,  10  mg.,is  necessary  to  kill  by 
the  mouth,  Roux  and  Yersin  endeavored  to  produce  immunity  to 
diphtheria  by  the  mouth,  but  were  unsuccessful. 

According  to  Fraser,  0.18  mg.  of  cobra  venom  constitutes  the 
minimum  fatal  dose  for  1  kilogram  of  rabbit.  The  guinea-pig  is  less 
susceptible,  and  the  kitten  still  less  so,  requiring  2  mg.  The  mini- 
mum fatal  dose  of  the  rattlesnake  venom,  per  kilogram  of  rabbit,  is 
placed  at  4  mg.  The  cobra  venom  is,  therefore,  16—20  times  more 
powerful  than  that  of  the  rattlesnake. 

Calmette  has  successfully  saved  rabbits  from  intoxication  by  venom 
by  injecting  in  a  circle,  at  a  distance  from  the  wound,  a  solution  of 
fresh  calcium  hypochlorite ;  in  the  case  of  man  an  injection  of 
20-30  c.c.  of  the  fresh  solution  obtained  by  diluting  a  1:12  solution 
(5  c.c.)  with  boiled  water  (45  c.c).  Mairet  and  Bosc  consider  this 
protection  by  hypochlorite  solution  as  due  to  a  direct  action  on  the 
venom  poison,  and  not  to  the  formation  of  antitoxin.  This  method 
of  treating  venom  bites  has  been  tried  with  success  in  Australia.  It 
should  be  noted  that  a  solution  of  hypochlorite  not  only  destroys  the 
poison  of  venom,  but  also  the  toxin  of  glanders  (Pench),  of  tetanus, 
and  diphtheria  (Roux). 

It  is  therefore  evident  that  a  striking  similarity  exists  in  the 
action  of  venom,  of  plant  albumoses,^  of  bacterial  toxins,  and  of 
enzymes.  The  similarity  is  strengthened  further  by  the  behavior  of 
these  poisons  to  heat  and  to  chemicals,  and  lastly  by  development  of 
antitoxic  substances  in  the  blood  of  animals  artificially  immunized 
against  these  toxins. 

Venoms  resemble  the  phytalbumoses  and  bacterial  toxins  in  their 
behavior  to  red  blood  cells.     Mitchell  and  Stewart,  in  1897,  showed 
that  these  poisons  agglutinate  and  then  dissolve  the  red  blood  cor- 
puscles.    The  work  of  Myers  on  cobra  lysins  has  been  mentioned  on 
^See  Cushny,  Archiv.  exp.  Path.  u.  Pharm.,  41,  447  (1898). 


480  CHEMISTRY  OF  THE  LEUCOMAINS. 

page  141.  Flexner  and  Noguchi^  have  recently  made  a  careful  study 
of  the  hemolytic  action  of  venoms.  Their  results  briefly  stated  are 
as  follows  :  Agglutination  of  corpuscles  occurs  rapidly  in  favorable 
solutions,  while  in  very  weak  ones  a  delay  of  some  minutes  up  to  one 
hour  may  be  noted.  Active  agglutination  takes  place  with  0.2  per 
cent,  solutions  of  venoms  but  the  maximal  results  are  given  by  0.5 
per  cent,  solutions.  The  corpuscles  of  the  rabbit  are  highly  sus- 
ceptible while  those  of  the  guinea-pig,  dog,  sheep,  swine  are  less  and 
in  about  the  order  given.  Hemolysis  follows  agglutination  and 
depends  on  the  strength  and  kind  of  venom  and  on  the  temperature. 
The  dog's  corpuscles  are  more  easily  hemolyzed  but  agglutinate  less 
readily  than  do  corpuscles  from  other  animals.  The  agglutinating 
power  of  venoms  is  destroyed  by  heating  at  75-80°  for  30  minutes. 
This  temperature  has  no  effect  on  the  hemolytic  power  which,  more- 
over, is  only  slightly  decreased  by  heating  at  100°  for  15  minutes. 
The  hemolytic  power  of  venoms  differs  :  cobra  is  most  active,  water- 
moccasin,  copper-head,  and  rattle-snake  are  less  in  the  order  named. 
The  different  mammalian  corpuscles  possess  a  varying  degree  of  sus- 
ceptibility. The  dog's  corpuscles  are  hemolyzed  most  rapidly  while 
those  of  sheep,  guinea-pig,  pig,  rabbit  and  ox  are  less  susceptible  in 
the  order  given.  The  hemolytic  effects  can  be  brought  out  usually 
with  0.2  per  cent,  solutions  of  the  venoms  but  in  the  case  of  the  ox 
a  0.5  per  cent,  solution  is  necessary.  Washed  corpuscles  are  agglu- 
tinated but  not  hemolyzed.  The  addition  of  serum,  however, 
promptly  induces  hemolysis.  Leucocytes  are  also  agglutinated  and 
hemolyzed  by  venoms.  The  agglutinating  principle  may  be  common 
to  both  the  red  and  white  corpuscles  while  the  dissolving  principle 
for  leucocytes  is  distinct  from  that  for  red  cells.  The  toxic  principle 
is  removed  by  nerve  cells  but  not  by  blood  cells.  Venom  destroys 
the  germicidal  properties  of  many  blood  sera  by  fixation  of  the  serum 
complements  by  the  venoms  which,  however,  have  no  action  upon 
the  intermediate  bodies  of  the  serum.  Antivenin  neutralizes  venom 
and  removes  both  the  hemolytic  and  antibacteriolytic  actions. 

The  blood  or  serum  of  the  common  turtle  (?)  (Bufo  vulgaris)  is  in 
1  c.c.  dose  toxic  to  frogs.  This  property  of  the  blood,  therefore,  is 
a  result,  as  in  the  case  of  the  viper  and  the  garter-snake,  of  the 
"  inner-secretion  "  of  toxic  glands  (Phisalix  and  Bertrand). 

Cloez  and  Gratiolet  in  1852  examined  the  poison  contained  in  the 
cutaneous  pustules  of  some  batrachians,  and  succeeded  in  extracting 
a  substance  which  gave  a  white  precipitate  with  mercuric  chlorid 
and  formed  a  platinum  double  salt.  Beyond  this  meagre  information 
very  little  is  known  in  regard  to  the  character  of  these  poisons, 
though  Zalesky,  in  1866,  announced  the  isolation  of  an  alkaloid  to 
which  he  assigned  the  formula  C3^Hg„N205,  and  which  he  named 
samandarin.      According  to  Dutartre  (1890),  this  base  is  a  leuco- 

J  Jovrn.  Exp.  Med..,  6,  277. 


Table  of  Leuoomains. 


Formula. 

Name. 

Discoverer. 

Source. 

Physiological 
Action. 

C5H5N, 

Adenin. 

Kossel. 

Nuclein  and 
urine. 

Poisonous. 

CsH^N^O 

Hypoxanthin. 

Scherer. 

Nuclein  and 
urine. 

Non-poison- 
ous, stimulant. 

C5H5N5O 

Guanin. 

Unger. 

Nuclein  and 
guano. 

Non-poi.son- 
ous,  stimulant. 

C,H,N50 

Epiguanin  or 
7-methyl  guanin. 

Kriiger  and 
Wolff. 

Urine. 

CjH.NA 

Xanthin. 

Marcet. 

Nuclein  and 
urine. 

Non-poison- 
ous, stimulant. 

CeHeN.O, 

Heteroxanthin  or 
7-methyl  xanthin. 

Salomon. 

Urine. 

Poisonous. 

1 -methyl  xanthin. 

Kriiger  and 

(1 

Salomon. 

3       " 
Paraxanthin  or  1-7 

Albanesi. 
Thudichnm, 

u 

C,HsNA 

(( 

Poisonous. 

di-methyl  xanthin. 

Salomon. 

Theophyllin  or  1-3 

Kossel. 

Tea-leaves  and 

di-methyl  xanthin. 

urine. 

Theobromin  or  3-7 

Woskresensky. 

Cocoa  and 

di-methyl  xanthin. 

urine. 

C^HsN.Oa 

Carnin. 

Weidel. 

Meat  extract; 
urine  (?) 

Non-poison- 
ous, stimulant. 

c,n,,^fi. 

Carnosin. 

Gulewitsch  and 
Amiradzibi. 

Meat  extract. 

^21^30^16^4 

Cytosin. 

Kossel  and 
Neumann. 

Thymus  nu- 
cleinic  acid. 

C.H.N^O^ 

Uracil. 

Ascoli. 

Nuclein. 

CsHjNj^^i 

Thymin  or  5- 
methyl  uracil. 

Kossel  and 
Neumann. 

(( 

CgHeN.O, 

Unnamed. 

Kutcher. 

Yeast. 

C,H«N30(?) 

Episarkin. 

Balke. 

Urine. 

C^H^NjOl?) 

Pseudoxanthin. 

Gautier. 

Muscle. 

CsHeN.OC?) 

Unnamed. 

Baumstark. 

Urine. 

CfiHi.NA 

Arginin. 

Schulze  and 
Steiger. 

Proteids. 

C«H9N,0, 

Histidin. 

Kossel. 

n 

C«Hi,N,0, 

Lysin. 

Drechsel. 

" 

CsHi^N.O, 

Ornithin. 

Jaffe. 

"  and  bird's 
urine. 

CsHuN, 

Gerontin. 

Grandis. 

Liver  of  dog. 

Poisonous. 

C,H,N(?) 

Spermin. 

Schreiner. 

Sperma. 

Non-poison- 

CloHgN 

Methyl  quinolin. 

Aid  rich  and 

Secretion  of 

ous. 
Non-poison- 

Jones. 

skunk. 

ous. 

CsHsN.O 

Cruso-creatinin. 

Gautier. 

Muscle. 

C,H,oN,0 

Xantho-creatinin. 

(( 

<< 

Poisonous, 

C9H„N;0, 

Amphicreatinin. 

" 

(( 

Ci,H,5N„05 

Unnamed. 

u 

It 

CiiHaiNioOj 

(4 

(( 

11 

C,H,2NA 

" 

Pouchet. 

Urine. 

C3H5NO, 

a 

a 

i( 

C3,H«,NA 

Samandarin. 

Zalesky. 

Salamander. 

Poisonous. 

VENOMS  OF  POISONOUS  SERPENTS.  481 

main,  and  eimilar  products,  but  with  different  physiological  action, 
are  to  be  found  in  other  batrachians,  as  the  toad,  triton  (?),  green  and 
red  frogs,  and  in  the  epidermis  of  some  fish.  According  to  Calmeil, 
the  poison  from  the  toad  contains  methyl  carbylamin  and  isocyan- 
acetic  acid.  According  to  Phisalix  and  Contejean,  the  blood  of  the 
salamander  possesses  antitoxic  action  with  reference  to  curare.  The 
salamander,  therefore,  is  naturally  immune,  and,  moreover,  its  blood 
will  protect  frogs  against  curara. 

The  recent  studies  of  Faust  ^  show  that  samandarin  acts  upon  the 
central  nervous  system.  The  chemical  nature  of  the  poison,  how- 
ever, remains  undetermined.  According  to  Phisalix  ^  the  salamander 
venom  loses  its  toxicity  on  drying ;  is  promptly  destroyed  at  100° 
and  even  at  50° ;  and  is  also  destroyed  by  alcohol.  The  venom 
altered  by  heating  to  60°  acts  as  a  vaccine  and  hence  resembles  the 
serum  of  eels. 

The  venom  of  bees  is  said  to  owe  its  toxicity  to  an  organic  base. 
That  of  the  hornet,  according  to  Phisalix,  immunizes  against  viper 
venom,  is  not  destroyed  at  120°,  is  soluble  in  alcohol  and  does  not 
contain  an  alkaloid. 

Phisalix'  has  also  shown  that  the  ventral  glands  of  myriapods 
(lulus  terrestris)  yield  a  toxic  secretion  which  is  not  affected  by 
heating  in  a  sealed  tube  at  100°  but  is  destroyed  at  120°.  The 
venom  is  supposed  to  contain  quinone. 

^Archiv.  ezp.  Path.  u.  Pharm.,  41,  229. 

2  Comptes  Bendus,  125,  121,  977  (1897). 

3  Comptes  Rendus,  131,  955,  1005,  1007.  , 


31 


CHAPTER   XVI. 

THE   AUTOGENOUS   DISEASES. 

All  living  things  are  composed  of  cells.  The  simplest  forms  of 
life  are  unicellular,  and  in  these  all  the  functions  of  life  devolve  upon 
the  single  cell ;  absorption,  secretion,  and  excretion  must  be  carried 
on  by  the  same  cell.  A  collection  of  unicellular  organisms  may  be 
compared  to  a  community  of  men  with  every  individual  his  own 
tailor,  shoemaker,  carpenter,  cook,  farmer,  gardener,  blacksmith,  etc. 
Nevertheless  it  is  true  that  in  communities  of  unicellular  organisms 
the  accumulation  of  their  own  secretions  and  excretions  impair  the 
growth  of  the  individual  and  finally  rob  it  of  its  life.  But  only  the 
lowest  forms  of  life  are  unicellular ;  all  others  are  multicellular.  In 
the  higher  animals  there  is  a  differentiation  not  only  in  the  size  and 
structure  of  the  cells,  but  in  the  duties  imposed  upon  them.  The 
body  of  man  may  be  compared  to  a  community  in  which  labor  has 
been  specialized.  Certain  groups  of  cells,  which  we  designate  by 
the  term  "organ,"  take  upon  themselves  the  task  of  doing  some 
special  line  of  work,  the  well  doing  of  which  is  essential  to  the  life, 
not  only  of  that  group,  but  of  other  groups  as  well,  or  of  the  body 
as  a  whole.  There  is  an  interdependence  among  the  various  organs. 
Certain  groups  of  cells  supply  the  fluids  or  juices  which  act  as  digest- 
ants,  and  among  these  there  is  again  a  division  of  labor.  The  sal- 
ivary glands  supply  a  fluid  which  partially  digests  the  starch  of  our 
food  ;  the  peptic  glands  supply  the  gastric  juice  which  does  the  pre- 
liminary work  in  digestion  of  the  proteids,  while  the  pancreatic  juice 
completes  the  digestion  of  the  starches,  begun  in  the  mouth,  of  the 
proteids  begun  in  the  stomach,  and  does  the  special  work  of  emulsi- 
fying fats.  Harm  results  to  the  individual  when  any  part  of  the 
digestive  processes  is  not  properly  carried  on,  and  even  some  of  the 
products  of  complete  digestion  are  harmful  when  permitted  to  enter 
the  circulation  unchanged.  The  peptons  must  be  converted  in  part  at 
least  into  serum  albumin  by  the  absorbing  mechanism  of  the  walls 
of  the  intestines,  and  while  ten  per  cent,  of  the  fat  of  the  food  is 
split  up  into  glycerin  and  fatty  acids  by  the  action  of  the  pancreatic 
juice,  a  much  smaller  per  cent,  enters  the  thoracic  duct  in  this  divided 
form.  The  food  taken  may  be  proper  both  in  quality  and  in  quan- 
tity and  the  digestive  juices  may  do  their  work  promptly  and  satis- 
factorily, but  if  the  absorbents  fail  to  perform  their  functions  properly, 
disease  results.  Again  it  may  happen  that  the  failure  lies  in  im- 
proper or  imperfect  assimilation,  and  the  result  becomes  equally  dis- 

482 


THE  AUTOGENOUS  DISEASES.  483 

astrous,  and  with  the  effects  of  uon-elimination  we  are  fairly  con- 
versant. Of  the  myriads  of  cells  in  the  healthy  human  body  there 
is  none  which  is  superfluous.  It  is  true  that  among  these  ultimate 
entities  of  existence  death  is  constantly  occurring,  but  in  health 
regeneration  goes  on  with  equal  rapidity  and  each  organ  continues  to 
do  its  daily  and  hourly  task.  The  microscope  has  made  us  familiar 
with  the  size  and  shape  of  the  various  cells  of  the  body,  and  pathol- 
ogists have  described  the  alterations  in  form  and  size  characteristic 
of  various  diseases  ;  but  we  must  remember  that  in  the  study  of  these 
ultimate  elements  of  life  there  are  other  things  besides  their  morpho- 
logical history  to  investigate.  They  are  endowed  with  life,  and  they, 
as  well  as  bacteria,  have  a  physiology  and  a  chemistry  which  we  but 
slightly  know.  Their  functions  are  influenced  beneficially  or  harm- 
fully, as  the  case  may  be,  by  their  environment.  They  grow  and 
perform  their  duties  properly  when  supplied  with  the  needed  pabu- 
lum, but  they  are  not  immune  to  poisonous  agents,  and  they  are  in- 
jured when  the  products  of  their  own  activity  accumulate  about  them. 

It  is  true,  without  exception  so  far  as  we  know,  that  the  excre- 
tions of  all  living  things,  plants  and  animals,  contain  substances 
which  are  poisonous  to  the  organisms  which  excrete  them.  A  man 
may  drink  only  chemically  pure  water,  eat  only  that  food  which  is 
free  from  all  adulterations,  breathe  nothing  but  the  purest  air,  free 
from  all  organic  matter,  both  living  and  dead,  and  yet  that  man's 
excretions  would  contain  poisons.  Where  do  these  poisons  originate? 
They  are  formed  within  the  body.  They  originate  in  the  metabolic 
changes  by  which  the  complex  organic  molecule  is  split  up  into 
simpler  compounds.  We  may  suppose — indeed  we  have  good  reason 
for  believing — that  the  proteid  molecule  has  certain  lines  of  cleavage 
along  which  it  breaks  when  certain  forces  are  applied,  and  that  the 
resulting  fragments  have  also  certain  lines  of  cleavage  along  which 
they  break  under  certain  influences,  and  so  on  until  the  end  products, 
urea,  ammonia,  water,  and  carbon  dioxid  are  reached ;  also  that 
some  of  these  intermediate  products  are  highly  poisonous  has  been 
positively  demonstrated.  The  fact  that  the  hydrocyanic  acid  mole- 
cule is  a  frequent  constituent  of  the  leucomains,  is  one  to  which  we 
have  already  called  attention.  We  know  that  chemical  composition 
is  an  indication  of  physiological  action,  and  the  intensely  poisonous 
character  of  some  of  the  leucomains  conforms  to  this  fact.  It  matters 
not  whether  the  proteid  molecule  be  broken  up  by  organized  ferments, 
bacteria,  or  by  the  unorganized  ferments  of  the  digestive  juices,  by 
the  cells  of  the  liver  or  by  those  still  unknown  agencies  which  induce 
metabolic  changes  in  all  the  tissues  —  in  all  cases  poisons  may  be 
formed.  These  poisons  will  differ  in  quantity  and  quality  according  to 
the  proteid  which  is  acted  upon  and  according  to  the  force  which  acts. 

It  is  not  our  intention  at  this  time  to  write  extensively  concerning 
the  autogenous  diseases.     We  desire  simply  to  call  attention  to  the 


484  THE  AUTOGENOUS  DISEASES. 

fact  which  has  been  illustrated  specifically  in  the  preceding  chapter, 
that  among  the  metabolic  products  of  the  animal  body  there  are  to 
be  found  substances  which  are  more  or  less  toxic  in  their  action. 
By  the  word  autogenous,  as  indicating  a  class  of  diseases,  we  under- 
stand that  in  these  disorders  the  maieries  morbi  is  a  product  of  some 
cell  of  the  body,  and  not,  as  in  the  case  of  the  infectious  diseases,  of 
cells  introduced  from  without  the  body.  Much  has  been  said  about 
auto-intoxication,  and  in  our  opinion  a  large  part  of  what  has  been 
said  upon  this  subject  is  without  sufficient  scientific  foundation. 
Auto-intoxication  is  a  word  now  frequently  used  to  cover  up  our 
ignorance,  and  the  time  has  not  yet  come  for  a  thorough  investiga- 
tion of  this  subject.  Indeed,  it  is  almost  impossible  in  a  given  case 
to  exclude  external  factors  which  influence  diseased  conditions. 
While  we  must  admit  that  poisons  are  formed  in  the  animal  body, 
it  is  not  always  easy — in  fact  we  may  say  it  is  never  easy  —  to  be 
certain  that  the  starting  point  does  not  lie  outside  the  body.  Some 
writers  have  mistaken  the  secondary  effects  of  ectogenous  toxins  for 
auto-intoxications.  As  an  illustration  of  this  we  may  mention  the 
attempt  that  has  been  made  to  classify  alcoholic  cirrhosis  under  the 
head  of  auto-intoxications.  The  explanation  which  has  been  offered 
by  those  who  have  advocated  this  classification  is  that  the  changes  in 
the  liver  are  not  due  to  the  direct  action  of  alcohol,  but  that  as  a  re- 
sult of  the  abuse  of  this  beverage  a  diseased  stomach  results,  and  on 
account  of  its  diseased  condition  permits  the  absorption  of  imper- 
fectly digested  substances,  to  the  direct  action  of  which  the  changes 
in  the  liver  and  other  organs  are  to  be  attributed.  According  to 
our  way  of  looking  at  it,  alcoholic  cirrhosis  is  due  either  directly  or 
indirectly  to  the  action  of  the  toxic  substance  which  is  introduced 
from  without,  and  certainly  should  not  be  classed  among  autogenous 
diseases.  Auto-infection  has  also  been  confounded  with  auto-intoxi- 
cation. 

These  two  processes  are  wholly  distinct,  and  a  clear  comprehension 
of  each  will  prevent  confusion.  The  term  auto-infection  is  properly 
applied  to  those  cases  in  which  the  virus  of  disease  is  carried  locally 
in  some  part  of  the  body,  and  is  distributed  from  this  locality  to 
other  parts  of  the  body.  Illustrations  of  auto-infection  are  numerous 
in  certain  diseases,  and  especially  in  tuberculosis.  An  individual 
with  pulmonary  tuberculosis  swallows  his  sputum  and  infects  his 
intestinal  walls.  Another  person  may  carry  for  years  a  colony  of 
tubercle  bacilli  in  his  lungs,  in  his  bones,  or  in  some  other  part  ot 
his  anatomy,  and  then  when  this  individual  is  exposed  to  conditions 
which  materially  lower  his  vitality,  the  bacilli  which  have  been  con- 
fined to  a  narrow  locality,  pass  beyond  the  bounds  to  which  they 
have  so  far  ])een  restricted,  and  invade  every  tissue,  leading  to  acute 
miliary  tuberculosis.  Post-mortem  examinations  have  shown  that 
one-third  or  more  of  all  men  have  latent  tuberculosis,  and  this  un- 


THE  AUTOGENOUS  DISEASES.  485 

doubtedly  is  the  explanation  of  the  frequently  observed  fact  that 
injury  to  some  part  of  the  body  leads  to  tubercular  lesions  in  that 
locality.  In  this  way  we  can  account  for  tuberculosis  of  the  hip 
joint,  of  the  spine,  and  of  the  meninges  of  the  brain.  These  illus- 
trations must  suffice  to  show  the  difference  between  auto-infection 
and  auto-intoxication. 

Some  writers  are  inclined  to  place  under  the  head  of  auto-intoxica- 
tions all  those  diseased  conditions  in  which  no  lesions  can  be  detected 
either  macroscopically  or  microscopically,  while  on  the  other  hand, 
they  exclude  from  the  list  of  autogenous  diseases  all  those  disorders 
which  are  accompanied  by  lesions.  This,  in  our  opinion,  is  plainly 
unscientific,  and,  indeed,  the  error  of  such  a  position  needs  only  to  be 
pointed  out  in  order  to  be  plainly  understood.  The  endogenous 
poisons  may  induce  lesions  which  are  quite  as  extensive  and  quite 
as  marked  as  those  which  result  from  the  ectogenous  toxins.  For 
instance,  recent  researches  have  shown  that  normal,  sterile  bile 
when  introduced  into  the  pancreatic  duct  and  brought  in  contact 
with  the  tissue  of  this  gland  causes  most  marked  necrotic  changes ; 
indeed,  it  is  doubtful  whether  or  not  we  have  among  the  bacterial 
toxins  any  substance  which  will  cause  more  marked  morphological 
changes  in  any  tissue  than  that  induced  by  normal  bile  when 
brought  in  contact  with  the  pancreas.  It  must  be  evident,  there- 
fore, that  we  cannot  exclude  from  the  autogenous  diseases  all  of 
those  which  show  tissue  changes.  Indeed,  it  is  probable  that  there 
is  no  disease  which  is  unaccompanied  by  morphological  changes  in 
the  cells  in  some  part  of  the  body.  These  alterations  may  be  so 
slight  that  they  have  escaped  detection,  but  it  certainly  is  true  that 
the  number  of  diseases  unaccompanied  by  morphological  changes 
grows  less  each  day. 

As  has  already  been  stated,  we  have  not  as  yet  sufficient  scientific 
data  to  enable  us  to  attempt  even  a  provisional  classification  of  the 
autogenous  diseases.  However  we  may  point  out  certain  facts  con- 
nected with  this  subject. 

1.  The  digestive  organs  may  but  imperfectly  perform  their  func- 
tion, and  the  products  of  their  incomplete  action  may  be  absorbed 
and  may  lead  to  more  or  less  disturbance  in  certain  organs  of  the 
body.  Moreover  in  such  a  case  as  this  every  part  of  the  body  will 
suffer  more  or  less  from  insufficient  nutrition  due  to  the  fact  that 
properly  prepared  pabulum  is  not  brought  within  reach  of  the  cellu- 
lar elements.  That  imperfectly  digested  proteids,  and  in  fact  certain 
proteids  wholly  undigested,  may  be  absorbed,  is  a  well  known  fact. 
To  what  extent  the  absorption  of  undigested  proteids  may  take 
place  in  the  animal  body  and  how  much  harm  can  be  wrought  in 
this  way,  we  are  not  able  to  say.  It  is  more  than  probable  that  the 
great  susceptibility  of  the  infant  to  bacterial  products  formed  in 
milk  is  due  to  the  fact  that  during  this  period  of  life  the  intestinal 


486  THE  AUTOGENOUS  DISEASES. 

walls  permit  the  passage  of  proteid  bodies,  to  which  the  same 
structure  in  the  adult  is  impervious.  When  peptons  and  albumoses 
are  injected  directly  into  the  blood  they  act  as  powerful  poisons. 
They  destroy  the  coagulability  of  the  blood,  lower  the  blood  pres- 
sure, and  in  large  quantities  cause  speedy  death.  The  lassitude  and 
depression  following  a  full  meal,  especially  one  rich  in  proteids,  is 
attributed  to  the  absorption  of  peptons,  but  so  far  there  is  no 
scientific  evidence  bearing  on  this  point.  Pepton  and  albumose  are 
frequently  found  in  the  urine,  but  whether  the  substances  found  in 
this  excretion  have  been  absorbed  from  the  intestinal  wall  or  have 
been  elaborated  elsewhere  in  the  animal  body,  no  one  knows. 

2.  That  certain  secretions  and  excretions  of  the  human  body  are 
poisonous  when  brought  in  contact  with  tissues  with  which  normally 
they  have  no  relation,  is  well  known.  We  have  already  referred  to 
the  action  of  normal  bile  when  brought  in  contact  with  the  pancreas, 
and  that  the  bile  acids  have  a  hemolytic  action  when  absorbed  into 
the  circulation  is  a  fact  which  has  long  been  known.  Why  it  is  that 
the  bile  has  a  destructive  action  upon  the  cells  of  the  pancreas  and 
no  such  effect  upon  the  cells  of  the  liver,  or  upon  the  structures  of 
the  gall  passages  and  intestines,  we  do  not  know.  It  is  rather  strange 
that  with  the  close  relation  between  the  pancreas  and  the  gall  blad- 
der that  the  contents  of  the  latter  do  not  more  frequently  reach  the 
former.  So  far  as  we  know  this  is  an  accident  which  very  seldom 
happens,  and  without  the  experimental  demonstration  which  has 
been  made  on  this  subject  it  would  be  quite  impossible  to  account  for 
the  alterations  observed  in  the  pancreas  in  the  few  instances  in  which 
this  has  happened. 

3.  It  is  the  function  of  certain  organs  of  the  body  to  prevent  the 
passage  of  certain  substances  into  the  general  circulation.  In  other 
words,  it  is  the  d  uty  of  certain  groups  of  cells  to  protect  other  com- 
munities from  harmful  agents.  The  rich  therapeutical  results  which 
have  followed  experimental  investigations  of  the  relation  of  the  thy- 
roid gland  to  myxedema  and  cretinism  are  illustrations  under  this 
head.  The  probabilities  are  that  myxedema  is  a  form  of  mucinsemia, 
and  that  the  introduction  of  an  excess  of  mucus  into  the  other  tissues 
is  prevented  by  the  normal  action  of  the  thyroid  gland.  This  pro- 
tective action  of  certain  glands  is  manifest  both  in  certain  internal 
and  external  secretions.  That  the  bile  consists  essentially  of  excre- 
mentitious  material  is  generally  believed  and  in  fact  has  been  demon- 
strated.    It  is  not  necessary  to  multiply  illustrations  of  this  kind. 

4.  That  the  undue  retention  of  excrementitious  substances  fre- 
quently leads  to  disturbances  of  health,  is  well  known.  The  ab- 
sorption of  effete  matter  from  the  intestines  and  the  retention  of 
substances  which  should  be  eliminated  by  the  kidneys  may  lead  to 
disastrous  results.  We  have  only  to  mention  as  an  illustration 
under  this  head  the  retention  of  urates  in  the  causation  of  gout,  and 


THE  AUTOGENOUS  DISEASES.  487 

the  absorption  of  bile  in  cases  of  obstructive  jaundice.  Our  studies 
of  the  leucomains  have  shown  that  small  amounts  of  substances  more 
or  less  toxic  are  constantly  being  formed  in  cellular  metabolism,  and 
the  undue  retention  of  these  leads  to  disease. 

5.  That  certain  cells  in  the  body  fail  to  adjust  themselves  to  gen- 
eral alterations  taking  place  in  other  organs  at  certain  periods  of  life, 
is  quite  evident.  So  true  is  this  that  the  physician  recognizes  the 
fact  that  there  are  certain  periods,  such  as  that  of  puberty  and  the 
climacteric,  which  are  accompanied  by  special  dangers  to  health  and 
even  to  life.  The  most  plausible  explanation  of  this  is  on  the  sup- 
position that  in  the  special  disturbances  of  certain  organs  other  parts 
of  the  body  fall  out  of  harmony,  and  the  parts  no  longer  work  to- 
gether smoothly. 

6.  Under  conditions  but  little  understood  at  present  certain  cells 
of  the  body  fail  to  utilize  certain  food-stuffs.  This  is  true,  for  in- 
stance, in  certain  forms  of  diabetes.  The  cells  which  are  accustomed 
to  absorb  and  utilize  the  sugars  find  themselves  unable  to  accomplish 
this  duty,  and  the  unused  sugar  acts  as  a  poison  to  other  tissues. 

7.  Active  poisons  are  sometimes  formed  by  certain  cells  in  the 
body.  In  this  way  we  account  for  the  presence  of  certain  of  the 
more  highly  toxic  leucomains  and  some  of  the  more  poisonous  acids, 
such  as  oxy-butyric,  and  some  of  the  poisonous  gases,  such  as  hydro- 
gen sulphid  and  methyl  mercaptan,  and  some  of  the  alkaloidal  bodies 
which  have  been  discussed  in  the  chapter  on  leucomains. 


INDEX. 


ACCIPENSEEIN,  429 
Acetvl-adenin,  361 
-cholin,  303 
-guanin,  383 
Addiment,  124 
Adenin,  341,  347 

-hvpoxantliin,  365 

-theobromin,  365 
Adenylic  acid,  347 
Adrenals,  bases  from,  473 
Adrenalin,  474 
Aerobic  bacteria,  30 
Agaricin,  293 

Agglutinable  substance,  154,  158,  161 
Agglutinate,  154 
Agglutination,  153,  177 

and  immunity,  179 

diagnosis  by,  159 

diflerentiation  of  bacteria  by,  160 

distinct  from  bacteriolysis,  123 

method  of  testings  159 

of  corpuscles,  115,  480 

pseudo-,  160 

theory  of,  161 
Agglutinins,  116,  137,  139,  152,  181,  184 

properties  of,  156 

relation  of  leucocytes  to,  157 
Albumins,  poisonous,  34 

precipitins  of,  115 
Albuminous  urine  precipitins,  117 
Albuminose,  434 

Albumoses  from  anthrax  bacillus,  49 
tubercle  bacillus,  80 
hog  cholera,  99 

immunity  from,  479 
Alcohol,  basic  substances  in,  231 
Alcoholic  fermentation,  bases  in,  280 
Aldehyde  collidin,  2-55 
Alexins,  109,  151,  167 

of  Bordet,  135 
Alexocytes,  111 
Alkaloids,  animal,  29 

interference     in    reactions     of,     by 
ptomains,  237 
Alkapton,  334 
Allantiasis,  201 
A  lloxuric  bases,  335 

bodies,  335 
Amanitin,  293 

Amido-valerianic  acid,  287,  438 
Amphi-creatin,  459 
Amyladenin,  364 

hypoxanthin,  375 


I  Amylamin,  33 

Amylic  alcohol,  impurities  in,  231 
Anaerobic  bacteria,  30 
Animal  chinoidin,  41 

coniin,  273 

parasites,  18 
Anthracen,  330 
Anthracin,  49 
Anthrax,  48 

albumose,  49 

bacillus,  products  of,  48 

action  of  pyocyanase  on,  175 
action  of  immune  serum  on,  179 

enzymes,  52 

immunity  to,  172 
by  enzymes,  175 

proteid,  49^  50 

ptomai'n,  49,  50 

susceptibility  to,  166,  167 

theories  of,  19,  48 

toxin,  51,  53 
Anti-alexins,  136 

-enzymes,  150 

-hemolytic  sera,  137,  141 

-infectious  sera,  179,  181,  184 

-lysin,  65,  130 

-spermotoxins,  148 

-staphylolysin,  88 

-toxic  sera,  179,  181 

theory  of  action  of,  181 
Antipepton,  424 
Antitoxin,  172 

artificial,  185,  179 

action  of,  181,  36,  71 

diphtheria,  71 

nature,  71,  183 

production,  130 

tetanus,  64,  67 

yenom,  478 
Antivenin,  141,  479 
Arbacin,  434 
Arginin,  423,  436 
Aromin,  464 
Arrow  poison,  68 
Asellin,  286 
Asiatic  cholera,  54 
see  vibrio 
Atmid  albumoses,  80 
Atropin-like  substances,  42,  244 
Auto-digestion,   basic   products   in,   265, 

272,  367,  416,  424 
Autogenous  diseases,  18,  482 
Auto-infection,  484 


489 


490 


INDEX. 


Auto-intoxications,    129,   291,   299,   334, 

484 
Autolysins,  129 
Auto-spermotoxins,  148 

-toxins,  148 
Azulmic  acid,  352 

BACILLUS  acidi  lactici,  86 
anthracis,  209,  283 

botulinus,  211 

bovis  morbificans,  208 

butyricus,  30 

coli,  97,  153,  215,  218,  220,  272 

enteritidis,  153,  207,  210 

piscicidus  agilis,  200 

pneumonife,  152,  204 

pyocyaneus,  86,  141,  150,  152,  166, 
179,  330 
immunity,  174 

subtilis,  86 

typhosus,  153 
Bacon,  poisonous,  206 
Bacteria,  action  of,  19 

in  summer  diarrhoeas,  91 

relation  of,  to  disease,  24 
Bacterial  cellular  proteids,  32 

diseases,  17 

intoxications,  24 

poisons,  definition  and  classification 
of,  29 
historical  sketch  of,  38 
foods  containing,  188 
of  the  infectious  diseases,  48 

products  in  toxicology,  237 

proteids,  31 

toxins,  22 
Bacteriolysis,  122,  174 
Batrachians,  poison  of,  480 
Baumstark's  base,  464 
Beef,  poisonous,  207 
Beer,  colchicin-like  substance  in,  247 
Bees,  poison  of,  481 
Benzol,  impurities  in,  231 
Benzoyl  adenin,  361 

chlorid  method,  267 

cholin,  304 

guanin,  383 
Benzyl  adenin,  362 

hypoxanthin,  374 
Bergmann  and  Schmiedeberg' s  method, 

236 
Betain,  304,  310 

aldehyde,  311 

homologues  of,  312 
Bilineurin,  293 
Blood,  germicidal  properties,  177 

hemolysis  and  agglutination,  123 

leucomains  in,  472 

maternal  and  fetal,  138 

precipitins,  119 
Blood-serum,  102 

antitoxic  and  bactericidal,  179 
protective  action  of  normal,  180 


Bocklisch's  base,  unnamed,  326 
Bois  bracelet,  198 
Bordet's  sensitizer,  161 
Botulinic  acid,  204 
Botulism,  193,  201 
Brain,  46,  294,  299,  348 

bases  in,  472 
Bread,  poisonous,  220 
Brieger*  s  bases,  unnamed,  313,  317,  325, 
327 

methods,  232 
Bromatotoxismus,  188 
Brom-adenin,  359 

-hypoxanthin,  373 

-guanin,  383 

-xanthin,  392 
Brouardel's  veratrin,  245 
Bujwid's  cholera-reaction,  59 
Butylamin,  253 
Butyric  acid,  438 
Butyro-cholin,  303 

pADAVERIC  alkaloids,  29  ' 
\J  coniin,  273 

Cadaverin,  47,  56,  271 

from  lysin,  444 
urine,  467 
Cafiein,  340,  405 
Caproylamin,  253 

Carbon  monoxide  in  expired  air,  463 
Carbonic  acid,  461 
Carbopyridic  bases,  457 
Carnin,  413 
Carnosin,  414 
Caseic  acid,  39,  203,  211 
Castor  bean,  47 
Causation  of  disease,  17 
Charcot  Neumann,  crystals  of,  450 
Cheese,  poisonous,  39,  211 

ptomains,  46,  47,  323 
Chemotaxis,  83,  86,  169 
Chicken  cholera,  100 

immunity  to,  172 

poisonous,  208 
Cholera,  54 

bases  in,  55,  56,  265 

-blue,  60 

Bujwid's  reaction,  59 

enzymes,  54 

immunity,  172 

-infantum,  61,  90 

precipitins,  113 

proteids  in,  56 

-red,  59 

serum,  122 

-stools,  39,  56,  271 

-toxin,  55,  57-59 
Cholin,  70,  293 

decompositions  of,  298 

group,  288 

constitution  of,  310 

in  Florence's  crystals,  450 
Chromatin,  430 


INDEX. 


491 


Cicuta  virosa,  239 
Classification  of  diseases,  18 

of  bacterial  products,  29 
Clupein,  429 

Codein-like  substances,  240 
Cod-liver  oil,  bases  from,  285,  286,  319 
Colchicin-like  substances,  246 
Colchicin,  Zeisel's  test  for,  246 
Coagulin,  184 
Collidin,  255,  256 
Collidone,  257 
Colon  toxin,  215 

Complement  body,  130,  134,  135,  136 
Complementoids,  133 
Coniin-like  substances,  43,  237 
Copper  in  foods,  191 
Copula,  136 
Coridin,  261 
Corindin,  259 
Corn,  poisonous,  223 
Corn-meal,  ptomai'ns  in,  45,  240 
Cornutin,  221 
Creatin,  56,  283,  378,  459 

relation  to  arginin,  437 
Creatinin,  70 

-group,  335,  454 
Cruso-creatinin,  457 
Cyclopterin,  429 
Cystin,  266,  269 
Cystinuria,  bases  in,  265,  268 
Cytase,  136 

Cytophil  group,  131,  182 
Cytosin,  415 
Cytotoxins,  147,  148 

DECONINCK'S  bases,  256,  260 
Delezinier's  base,  263 
Delphinin-like  substances,  245 
Derris,  198 
Desmon,  136 
Deutero-albumose,  49 

toxins,  35,  65 
Diamins,  263,  271,  272,  472 
Di-amido  caproic  acid,  444 

valerianic  acid,  438 
Diaminuria,  265,  268 
Diarrhoea,  petromyzon,  197 
Diarrhoeas  of  infancy,  90 
Diethylamin,  252 
Digitalin-like  substances,  43,  244 
Dihydrolutidin,  254 
Dimethylamin,  249 

-guanin,  383 

-hypoxanthin,  374 

-xanthin,  339,  400,  404 
Diphtheria,  68 

bacillus,  frequency  of,  25 
products  of,  47,  68 

immunity  to, 

pathological  changes  in,  75 

serum,  preparation  of,  165 

toxalbumin,  70 

toxin,  68,  72 


Diphtheria,  Ehrlicli's  views,  35,  71 
Diseases,  classification  of,  17,  18 
Dragendorff's  method,  232 
Drechsel's  reaction,  353,  408 

UBEKTH'S  bacillus,  products  of,  30 

Vj     Echidnase,  477 

Echidnin,  477 

Echidnotoxin,  477 

Eel  serum,  antitoxic  action,  478 

hemolytic  action,  129,  137 
precipitin,  117 
Egg  albumin  precipitin,  115 
Ehrlich's  view  of  diphtheria  toxin,  35,  71 

theory  of  hemolysis,  129 
immunity,  181 
Emmerich's  theory  of  immunity,  174 
Enzymes,  anti,  150 

in  disease,  21 
anthrax,  52 
diphtheria,  70 
germinating  plants,  423 
hog  cholera,  99 

action  on  agglutinins,  156 

bacteriolytic  action,  174 

relation  to  immunity,  174 
Epiguanin,  386 
Epinephrin,  474 
Episarkin,  420 
Epitheliolysins,  142 
Ergot,  221,  250 
Ergotismus,  220 
Ethyl  adenin,  364 

guanin,  384 

-hypoxanthin,  375 

-amin,  251 
Ethylenediamin,  47 

-imin,  450,  451 
Ethylidenediamin,  263 
Etiology  of  bacterial  diseases,?17 
Expired  air,  leucomains  in,  461 
Examination  of  poisonous  foods,  226 

FATTY  acids,  52 
Faeces,  poisons  in,  272 
Fish,  canned,  199,  200 
fermented,  249 
poisonous,  193 
ptomai'ns,  47,  198 
Florence's  crystals,  450 
Food  poisoning,  188 

examination  in,  227 
Friedlsender"  s  bacillus,  33 
Fugu,  195 
Fungous  diseases,  17 

GADININ,  47,  315 
Gaduin,  320 
Galactin,  473 
Galactotoxismus,  215 
Gautier's  leucomains,  459,  460 
Gautier  and  Etard's  bases,  286 
methods,  233 


492 


INDEX. 


Gautier  and  Etard's  extraction  of  leuco- 

mains,  455 
Gelatin,  hexon  bases  from,  426 

ptomai'ns  from,  47 
Germicidal  action  of  serum,  102 
Gerontin,  449 
Glanders,  101,  262 
Globin,  435 
Globulin,  germicidal  properties  of,  104 

Halliburton's,  104 

precipitins,  115 
Glucosins,  281 

Glycogen  in  tubercle  bacillus,  82 
Gonococcus,  89 
Gonotoxin,  89 
Goose  grease,  poisonous,  209 

poisonous,  238 
Gram's  bases,  326 
Griffith's  bases,  261 
Guanidin,  341,  380 

from  arginin,  438 

butyric  acid,  438 
Guanin,"  340,  376 

constitution,  340 

preparation  of,  384 
Guareschi's  base,  323 

and  Mosso's  bases,  326 

HALICHTHYTOXIN,  198 
Ham,  poisonous,  210 
Haptophorous   group,   36,   65,   128,   129, 

130,  182 
Hemitoxin,  35 
Hemoglobin,  435 
Hemoglobinuria,  136 

Hemolytic  action  of  stapliylolysin,  88 

of  tetanolysin ,  63 

of  venoms,  480 
Hemolysis,  123,  134,  137 
Hemotropic  group,  128 
Heteroagglutinins,  140 
Heterolvsins,  129,  140 
Heteroxanthin,  339,  397 
Hexamethylenediamin,  279 
Hexon  bases,  335,  422 
action  of,  433 
in  proteids,  426 
method  of  separation,  427 
Hexylamin,  253 
Histidin,  426,  442 
Histon,  428.  434 

action  of,  432 
Historical  sketch  of  bacterial  poisons,  38 
Hog-cholera,  99,  281 
Homologous  sera,  152 
Honey,  toxic,  197 
Hornet,  poison,  481 
Hydrocollidin,  258 
Hydrocoridin,  261 
Hydrogen  sulphid,  52,  57 
Hydrolutidin,  254 
Hydrophobia,  173,  186 
Hyoscyamin-like  substances,  42,  244 


Hypoxanthin,  341,  365 

TCE-CREAM,  poisonous,  219 

1     Ichneumon,  immunity  to  venom,  170, 

478 
Ichthyotoxismus,  193 
Idio-isolysins,  139 

iso-agglutinins,  139 
Immune  body,  124 
Immunity,  163 

active,  165 

and  agglutination,  179 

artificial  or  acquired,  165,  171 

bacterial,  179 

inherited,  165 

methods  of  inducing,  171 

natural,  165 

passive,  165 

produced  bv  non-bacterial  products, 
173 

r61e  of  phagocytes,  168 
Indol,  59,  241 
Indoxyl,  453 
Infection,  18 

and  intoxication,  bacterial,  164 
Infectious  diseases,  18 

definition  of,  23 
how  produced,  23 
poisons  of,  48 
Intermediary  body,  128,  130,   135,  136, 

151 
Intoxications,  18,  24 
Intracellular  toxins,  166 
Iso-amylamin,  253 

-agglutinins,  139 
Isolysins,  129 

idio-,  139 
Iso-muscarin,  311 

-propylamin,  253 

TEQUIRITY,  47 

KAKKE,  196 
Koch's  rules,  24 
Kreotoxismus,  201 
Kynurenic  acid,  453 

T  ACTOCHOLIN,  303 

1j    Lactochrome,  473 

Lactoserum,  115 

Lafon's  reagent,  243 

Lathyrismus,  222 

Lecithin,  decomposition  of,  296,  305 

preparation  of,  296 
Lepierre's  base  from  cheese,  323 
Leucin,  33,  49,  57,  101,  255 

homologues  of,  317 
Leucinijnid,  452 
Leucocidin,  88,  109 
Leucolysin,  149 
LeucocythiBmia,  urine  in,  348 
Leucocytosis,  87 


INDEX. 


493 


Leucomai'ns,  29 

chemistry  of,  332 

tables  of,  481 
Lutidin,  254 
Lycin,  305 
Lysatinin,  425 
Lvsin,  425,  444 
Lysins,  122,  181 
Lysuric  acid,  446 

MACROPHAGES,  168 
Malignant  oedema,  101 
Maidismus,  223 
Mallein,  101 

Meal  and  bread,  poisonous,  220,  222,  223 
Meat,  poisonous,  47,  201 
Mechanical  interference,  theory  of,  21 
Meissner'  s  base,  465 
Methyladenin,  363 

a'min,  248 

betain,  305 

carbylamin,  481 

guanin,  383,  386 

guanidin,  47,  56,  100,  282,  284 

hydantoi'n,  460 

hypoxanthin,  375 

quinolin,  452 

uramin,  283 

xanthin,_  339,  394,  395,  397 
Milk,  agglutinins,  156 

bacteria,  94 

leucomai'ns  in,  473 

poisonous,  215 

precipitins,  114 

sera,  action  of,  143 
Monamins,  248 
Mongoose,  170,  478 
Morin'  s  base,  280 
Morphin-like  substances,  240 
Morrhuic  acid,  319 
Morrhuin,  285 
Morvin,  101 
Murexid,  392 
Muscarin,  47,  307,  311 
Mussel,  poisonous,  189 
Mycoderma  aceti,  30 
Mycoprotein,  33 
Mydatoxin,  47,  311 

isomer  of,  313 
Mydalein,  47,  324 
Mydin,  47,  257,  287 
Myriapods,  poison  of,  481 
Mytilotoxin,  47,  191,  313 
Mytilotoxismus,  189 

NARCOTIC  substance  of  Panum,  41 
Nencki's  base,  255 
Nephrotoxin,  89,  143 
Neubauer's  method,  407 
Neuridin,  47,  209,  276 
Neurin,  47,  288,  310 
Neurotic  diseases,  19 
Nicotin-like  substances,  43,  239 


Nicotinic  acid,  258 
Nuclease,  175,  177 
Nuclein  bases,  335 
Nucleins,  98,  334  _ 

germicidal  action,  109,  110 
metabolism  of,  336 
Nucleinic  acid,  109,  110,  429 

in  phagocytes,  169 

metabolism  of,  343 
Nucleohiston,  350,  434 
Nucleosin,  416 

ARNITHIN,  437,  446 

U     Ornithuric  acid,  437 

Oser's  base,  286 

Ovasera,  115 

Oxalate  plasma,  agglutination  by,  157 

Oxy-betains,  320 

-cholin,  308 

-neurin,  304 
Oxygenated  bases,  248 
Oysters,  poisonous,  192 

PACHYRRHIZID,  198 
Panum' s  narcotic  substance,  41 
putrid  poison,  40 
Paraffin  oil,  bases  in,  281 
Parareducin,  464 
Paraxanthin,  339,  400 
Parvolin,  259 
Pathoamins,  465 
Pellagra,  223 
Pellagrocein,  240 
Pentamethylenediamin,  272 
Peptonizing  bacteria  from  milk,  95,  96 
Pepton,  poisonous  nature  of,  96 

sera,  116 
Peptotoxin,  328 
Petroleum,  bases  in,  281 
Petromyzon,  197 
Pfeiffer's  phenomenon,  122 
Phagocytes,  168 
Phagolysis,  173 
Phenol,  33,  243 
Phenyl  adenin,  364 

-alanin,  256 

-ethylamin,  256 
Phlogosin,  85,  327  _ 
Phosphorus-containing  substances,  44 
Phylocytase,  136 
Phytalbumose,  476 
Piperazin,  451 

Piperidin,  synthesis  of,  272,  442 
Plague  precipitins,  113 
PlasmolysiSj  170 

Pneumonia,  chemical   products   in,  100, 
283 

immunity  to,  100 
Poisonous  arrows,  68 

foods,  188 

examination  of,  226 
Pork,  202,  204 
Pouchet's  bases,  320,  323 


494 


INDEX. 


Pouchet's  bases  in  urine,  464 
Precipitins,  113 
Propylamin,  43_,  252 
Propionyl  guanin,  383 
Protalbumose,  49 
Protamin,  81,  428 

separation  of,  430 

germicidal  action  of,  432 

physiological  action  of,  433 
Proteids,  bacterial.  31 

poison,  42 

vulgaris,  87,  91,  263 
Protons,  429,  431 
Prototoxins,  35,  65 
Prototoxoid,  65 
Protozoal  diseases,  18 
Pseudo-xantliin,  422 
Ptomain  poisoning,  226 
Ptomains,  chemistry  of,  248 

definition  of,  29 

methods  of  extraction  of,  230 

table  of,  330 
Ptomatins,  29 
Ptomatropin,  244 
Puerperal  fever,  101 
Purin,  336,  346 

bases,  separation  of,  407 

group,  334,  336 
action  of,  345 

metabolism  of,  342,  344,  406 
structure  of,  337 
Pus  organisms,  85 

bases  from,  349 
Putrefactive  alkaloids,  29 
Putrescin,  47,  56,  265,  438 

in  urine,  467 
Putrid  material,  effects  of,  38 

poison  of  Panum,  40,  42 
Pyocyanin,  86,  152,  330 
Pyocyanase,  175 

action  on  bacteria,  176 
Pyogenetic  proteids,  83,  86,  90 
Pyoxanthose,  330 
Pyrazin,  418 
Pyridazin,  418 
Pyridin,  281,  319 
Pyrimidin,  417 

group,  334,  415 

relation  to  uric  acid,  419 
Pyrrolidin  carbonic  acid,  448 

QUINOLIN,  453 
Quinone,  481 

RABBIT  septicemia.  100 
Receptors,  129,  130 
Eeducin,  463 
Rennet,  anti-,  150 
Eoussin's  test  for  nicotin,  237 

OALAMANDER,  480 

O      poLson,  480 

Saliva,  leucomains  in,  471 


Salkowski's  base,  287 

Salmin,  429 

Salmon,  199 

Samandarin,  480 

Saprin,  47,  278 

Sarcin,  365 

Sardines,  poisonous,  200 

Sarkin,  365 

Sausage,  poisonous,  39,  43,  201,  249 

Scombrin,  429 

Scombron,  434 

Sebacic  acid,  39,  204,  211 

Selmi's  method,  43 

Sepsin,  41 

Septicemia,  24 

of  rabbits,  100 
Septicin,  253 
Serum,  germicidal  action,  102,  169 

agglutination  by  normal,  153,  154 

precipitins,  118 

-therapy,  47 
Siguatera,  193 
Silurin,  429 
Sinkalin,  289 
Sitotoxismus,  220 
Skatol,  242,  453 
Skin  burns,  329,  466 
Skunk  secretion,  452 
Small-pox,  171 
Spasmotoxin,  61,  254 
Spermatolysins,  145 
Spermatoxins,  145 
Spermatozoa,  agglutination  of,  146 

composition  of,  429,  435 
Spermin,  450 
Sphacelinic  acid,  221 
Spleen,  leucomains  in,  473 
Staphylococcus  pyog.  aureus,  86,  88 

bases  from,  327 

hemolytic  action,  88 

flavus,  210 
Staphylolysin,  88 
Staphylotoxin,  88 

pathological  changes  by,  89 
Stas-Otto  method,  231 
StreiJtococcus,  91,  249,  250,  255 

immunity  to,  173 
Strychnin-Uke  substances,  239 
Sturin,  429 

Substance  sensibilatrice,  135 
Succinic  acid,  438 
Sucholotoxin,  99 
Summer  diarrhceas'of  infancy,  90 
Suppuration,  85 
Suprarenin,  474 
Susotoxin,  99,  280 
Swine-plague,  immunity  to,  180 
Symptomatic  anthrax,  209 

TETANIN,  61,  320 
1      Tetanolysin,  36,  63 
Tetanospasmin,  36,  63 
Tetanotoxin,  61,  254 


WDEX. 


495 


Tetanus,  61 

antitoxin,  65,  66 

bacillus,  products  of,  34,  47,  61,  254, 

313,  320 
toxins,  34,  61 

hemolytic  action,  63,  64 
elimination,  66 
action  on  nerve  cells,  66 
Tetramethylenediamin,  267 
Tetramethyl-putrescin,  269 
Tetrodon,  195 
Tetrodonin,  196 
Thein,  see  caffein 
Theobromin,  339,  404 
Theophyllin,  337,  339,  404 
Thymin,  416 

Toxalbumins,  30,  50,_  61,  70,  97 
Toxicology  of  bacterial  products,  237 
Toxins,  22,  29,  33,  35 

conversion  into  antitoxin,  185 
minimum  fatal  dose,  72 
natural  immunity  to,  170,  171 
pathological  changes  by,  73 
Toxoid,  35,  65,  133 
immunity,  37 
of  the  staphylococcus,  88 
Toxons,  35,  65,  72 
Toxopeptons,  57 
Toxophil  chains,  182 
Toxophorous  group,  36,  65,  130,  182 
Traumatic  disease,  18 
Trichiniasis,  201 
Triethylamin,  252 
Trigonellin,  305 
Trimethylamin,  47,  250 

xanthin,  340,  405 
Trimethylenediamin,  56,  264 
Tritotoxins,  35,  65 
Tubercle  bacillus,  products  of,  77 
chemotatic  action,  83 
fatty  products,  79 
nucleinic  acid,  81 
proteid  products,  80 
protamin,  81 
ptomains,  82 
Tuberculin,  77,  83 

action  of,  84,  139 
Tuberculinic  acid,  81 
Tuberculosamin,  81,  429 
Tuberculosis,  76 
Turtle,  480_ 
Turkey,  poisonous,  210 
Typhoid  bacillus,  97 

agglutination  of,  113 
fever,  97,  165 

serum  agglutination  in,  155,  159 
-like  bacilli,  210 
Typhotoxin,  47,  97,  316 

isomer  of,  317 
Tyrosin,  49,  57,  101,  255 
Tyrotoxicon,  46,  213,  323 

in  summer  diarrhoea,  94 
Tyrotoxismus,  211 


UNDETERMINED  leucomains,  461 

L)  ptomains,  323-330 

Uracil,  417,  418 

Urea,  as  cleavage  product,  437,  459 
Uric  acid,  333,  334 

deposits  of,  344 

group  of  leucomai'ns,  334 

metabolism  of,  343 

purin  bases  from,  337 

reduction  of,  337 

separation  of,   from  xanthin  bases, 
412 

synthesis  of,  419 
Urine,  agglutination  by,  155 

diphtheria  toxin  in,  70,  468 

leucomai'ns  in,  250,  389,  463 

toxicity  of,  468 
Urochrome,  464 
Urotheobromin,  400 
Urotoxin,  465 
Urotoxy,  469 
Urotoxic  coefficient,  469 
Uschinsky's  medium,  34 

T7ALERIANIC  acid,  33 

V      Valero-cholin,  303 

Veal,  poisonous,  205,  209 

Vegetable  foods,  poisoning  by,  220 

Vetch,  poisonous,  222 

Venoms,  47,  474 

agglutinating  action,  480 
artificial  immunity  to,  172,  476 
hemolytic  action,  137,  141,  479 
natural  immunity  to,  170 

Veratin-like  substances,  245,  263 

Vernin,  378 

Vibrio   cholerse  asiaticse,   153,  170,   248, 
264 
Metchnikovi,  152 

Vinylamin,  143 

Vitiated   air,    effects   of   inhalation     of, 
461 

WEIDEL'S  reaction,  352,  370 
White  liquefying  bacterium,  prod- 
ucts of,  94 
Widal's  reaction,  154 

XANTHIN,  339,  388 
bases,  335 
constitution  of,  339 
isolation  of,  410 
reaction,  352,  370,  392 
Xantho-creatinin,  457 

yEAST,  30,  33 

1      auto-digestion  of,  348 

decomposition  of,  326 

nuclein,  100 

yEISEL'S  test  for  colchicin,  246 


\'hO'2_ 


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